ATCC Cell Culture Technical Resource: in Partnership With

ATCC Cell Culture
Technical Resource
in partnership with
ATCC general accessioning process
The general ATCC cell line accessioning scheme encompasses a series of tests which confirm the identity of a cell line
and ensures that it is free of contamination.
A systematic seed-stock cell-banking method is used to produce virtually identical distribution lots, ensuring consistent
materials for every order.
ATCC cell lines and hybridomas are provided with comprehensive and repeated authentication and contamination
checking – starting with the culture derived from the depositor’s ampoule and continuing through the production of
vials for distribution – ensuring that delivered materials meet the highest standards.
Verification by depositor
Starter culture Token freeze Seed stock Distribution
• Contamination checks • Contamination checks • Contamination checks
• Species verification • Species verification • Species verification
• Post-freeze recovery • Post-freeze recovery • Post-freeze recovery
• Post-freeze viability • Post-freeze viability • Post-freeze viability
• Growth curve • Characterisation tests • Characterisation tests
• Characterisation tests
Figure 1 The general ATCC accessioning process includes many tests that are repeated at every stage to provide cell line identity verification and unsurpassed
quality-testing for all bioproduction runs.
Authenticated Robust growth

Low passage Consistent cultures
Contaminant free Reproducible results
ATCC authentication – Verifying cell lines
Experimental success has been shown to correspond

directly to the quality and condition of the cell lines used.
Cells that are kept too long in culture and are not periodically
tested for genotypic or phenotypic stability may no longer
be reliable models of the original source material.
To maintain high cell culture standards and ensure reliable,

reproducible results, the use of authenticated and
quality-tested cell lines from a recognised cell bank is
highly recommended.
ATCC authenticates cell lines routinely with the

following tests:
I Short tandem repeat (STR) profiling establishes a DNA
fingerprint for human cell lines.

The pattern of repeats results in a unique STR identity
profile for each cell line analysed. STR analysis is critical
for verifying the identity of human cell lines and is
ATCC STR profiling uses multiplex PCR to simultaneously performed for each distribution lot. The results are
amplify the amelogenin gene and eight of the most compared to the baseline profile of the token stock
informative polymorphic markers in the human genome. derived from the depositor.
Figure 2 I STR profile of two unrelated cell lines. Top:

KU812E (ATCC® CRL-2100™). Bottom: MRC-5 (ATCC®
CCL-171™). Amplicons are generated using Promega
PowerPlex® 1.2 system, separated by electrophoresis
and analysed using Genotyper® 2.0 software from
Applied Biosystems.
“Evidence suggests that up to one-third of tumor cell

lines being used in scientific research are affected by
inter- or intraspecies cross-contamination or have been
wrongly identified, thereby rendering many of the
conclusions doubtful if not completely invalid.”
Lancet Oncology, vol. 2, July 2001, p. 393
ATCC authentication – Verifying cell lines
I Cell morphology is monitored throughout all ATCC I Karyotyping is performed to identify the species
processes. as well as variation within the cell line.
Cellular morphology can vary between lines Karyotyping is a basic and indispensable test
depending on the health of the cells and, in some performed routinely to determine if the line has
cases, the differentiation state — a critical property maintained a stable genotype. Karyotyping is
in certain assays. Morphology can change with performed on many ATCC classic cell lines and
plating density as well as with different media and all embryonic stem cell lines.
sera combinations. Morphologies of cells grown at
low and high densities at ATCC are recorded and
used routinely to check cell lines during
accessioning and bioproduction.
Figure 3
ATCC® CCL-1™ at high
cell density
Figure 4
ATCC® CCL-1™ at low Figure 5 ATCC® CRL-4001™ Giemsa-banding on distribution (top) and
cell density seed (bottom) stocks
The ATCC COI assay is used to reliably determine the species

of a cell line.
The use of cytochrome C oxidase I (COI) testing at ATCC replaces
isoenzymology in determining the true species of a cell line. The cytochrome
C oxidase I (COI) is conserved genetic material found in the mitochondria
among closely realted species and across diverse phyla in the animal
kingdom.32* Based on the species-to-species sequence variability of the
COI gene, ATCC scientists developed a PCR-based speciation assay by
designing unique primer pairs that recognise only a specific species and
Figure 6 Amplified fragments were detected by
producs amplicons in a multiplex PCR reaction with sizes no less than 20
base pairs apart.29 The ATCC COI assay is capable of distinguishing cell ethidium bromide staining on a 4% agarose gel. Lane
lines of pig, human, cat, Chinese hamster, Rhesus monkey, sheep, horse, 1 shows the 100 bp ladder. Lane 2 shows the multiplex
performance of oligonucleotide pairs specific for the
African green monkey, rat, dog, mouse, rabbit, goat and cow origin. When following 14 species: pig, human, cat, Chinese
the species of a cell line remains in question 65bp ‘barcode’ region of the hamster, Rhesus monkey, sheep, horse, African green
monkey, rat, dog, mouse, rabbit, goat and bovine. The
COI gene is sequenced for verifiction purposes. template for the reactions consisted of 0.5-1.0 ng
* For more information on the Barcode of Life initiative, please see: www.barcodinglife.com
mixed DNA contributed from all the species with
primers in the master mix.
Technical Information
Contents
6 Getting started with an ATCC cell line
7 Cell growth and propagation
13 Complete growth media
18 Culture vessels and surfaces
20 Cryopreservation
23 Contamination
24 Biosafety
25 Glossary
28 Formulations of media not available

from ATCC
30 References
32 Why do cell lines need to be authenticated?
34 Disclaimers
35 Material Transfer Agreement
38 Notes
39 The LGC Standards – ATCC Partnership

Getting started with an ATCC cell line
ATCC cell lines and hybridomas are shipped frozen on dry ice NOTE: Some cell lines, such as hybridomas, take several days
in cryopreservation vials or as growing cultures in flasks at before fully recovering from cryopreservation. Some hybridomas
ambient temperature. Upon receipt of frozen cells, it is important have poor viability the first day in culture and will generate
to immediately revive them by thawing and removing the DMSO cellular debris. After this point, the cells will begin to recover
and placing them into culture. If this is not possible, store the cells and enter exponential growth.
in liquid nitrogen vapour (below -130°C). Do not store frozen cells
at temperatures above -130°C as their viability will decline rapidly. It is important to refer to the Product Information Sheet provided
with your culture; this lists specific growth requirements for
Product Information Sheet individual cell lines.
ATCC cell lines come with a Product Information Sheet that Processing flask cultures
contains detailed information for handling the cells. An
abbreviated version may be found at the ATCC website or Some ATCC cell lines, primarily those from the NBL collection
contact our Technical Service Department to request a copy. are shipped as growing cultures in culture vessels. These
The Product Information Sheet also contains batch-specific vessels are seeded with cells, incubated to ensure cell growth
information such as the number of cells per vial, the and then filled completely with medium for shipping.
recommended split or subcultivation ratio, and the passage
number when known. Upon receiving a flask culture, visually examine the medium for
macroscopic evidence of microbial contamination. This
Preparation of medium includes unusual pH shifts (yellow or purple colour from the
phenol red), turbidity, or particles. With an inverted microscope
Prepare for reviving cell lines by assembling the appropriate at low power (100x) check the medium for evidence of microbial
medium, serum, and additional reagents required for growth. contamination as well as the morphology of the cells. See page
Many of these products are available from ATCC and can be 7 for more details on examining cell cultures.
If the cells are attached and growing in a monolayer:

ordered with the cell lines (see page 12 for details). These are
the same reagents used by ATCC for cell growth and
preservation.
1. Aseptically remove all but 5 to 10 ml of the shipping medium.
NOTE: While most cell lines can replicate in more than one The shipping medium can be saved for reuse and should
culture medium, their characteristics may alter when the medium be stored at 4°C.
is changed. For this reason, starting cell cultures in the same 2. Incubate the flask at the temperature and CO2 concentration
medium used by ATCC is recommended for the best results recommended on the Product Information Sheet (37°C with
(see the Product Information Sheet and ATCC website). For 5% CO2 for most cell lines) until the cells are subcultured.
details on adapting a cell line to a new medium, see page 7.
If the cells are not attached or are growing in suspension:
Initiating frozen cultures
1. Aseptically transfer the entire contents of the flask to a
1. Prepare a culture vessel so that it contains the recommended
Centrifuge at 125 x g for 5 to 10 minutes.
centrifuge tube.
volume of the appropriate culture medium as listed on the 2.
Product Information Sheet, equilibrated for temperature 3. Remove all but 10 ml of the shipping medium supernatant
and pH (CO2). and resuspend the cells. Store the remainder of this medium
2. Thaw the vial by gentle agitation in a water bath at 37°C at 4°C for later use.
or the normal growth temperature for that cell line. Thawing 4. Aseptically transfer the resuspended cells to a 25 cm2 flask
should be rapid, approximately 2 minutes or until ice crystals or 75 cm2 flask, depending upon the cell line (see the
have melted. Product Information Sheet).
3. Remove the vial from the water bath and decontaminate it 5. Incubate the cells at the temperature and CO2 concentration
by dipping in or spraying with 70% ethanol. Follow strict recommended on the Product Information Sheet until cells
aseptic conditions in a laminar flow tissue culture hood for are subcultured.
all further manipulations.
4. Unscrew the top of the vial and transfer the contents to a
sterile centrifuge tube containing 9 ml of the recommended
gentle centrifugation (10 minutes at 125 x g). Discard the

medium. Remove the cryoprotectant agent (DMSO) by
supernatant, and resuspend the cells in 1 or 2 ml of complete

growth medium. Transfer the cell suspension into the
culture vessel containing the complete growth medium and
mix thoroughly by gentle rocking.
5. Examine the cultures after 24 hours. Subculture as needed.
These products are for laboratory research use only. Not intended for use in humans, animals or for diagnostics.
6 Order online at www.lgcstandards-atcc.org or contact your local LGC Standards office. See back cover for details.
Cell growth and propagation
Most cell lines begin as primary cultures originating from a piece To ensure viability, genetic stability, and phenotypic stability, cell
of minced or enzyme-dispersed tissue. Primary cultures, as lines need to be maintained in the exponential phase. This
mixtures of several cell types, retain the characteristics of their means that they need to be subcultured on a regular basis before
source tissue. they enter the stationary growth phase, before a monolayer
becomes 100% confluent or before a suspension reaches its
After a period of time, primary cultures will reach confluency, maximum recommended cell density. Generating a growth
the state when all available space of the culture vessel is curve for each cell line is useful to determine the growth
covered due to cellular expansion. At this point, the culture will characteristics of the cell line (see Figure 1).
need to be disaggregated (usually with proteolytic enzymes like
trypsin) into individual cells and subcultured (split, passaged, or For detailed information on the growth and propagation of any
transferred). Following this first passage, the culture is generally ATCC cell line, see the specific cell line Product Information
referred to as a cell line. With each subsequent subculture, the Sheet which is included with every shipment. An abbreviated
cellular population becomes more homogeneous as the faster version may also be found on the ATCC website, or contact
growing cells predominate. Cells with desired properties can Technical Services to have one sent to you. The Product
also be selected out of the culture by cloning. Information Sheet contains valuable information about growth
medium, subculturing procedure, split ratio, and any
Diploid cell lines rarely progress beyond a few population requirements for feeding the culture between passages.
doublings.They have a finite replicative capacity and begin to
slow down and eventually stop dividing after 20 to 80 population Passage number and population doubling level
doublings.1 Recent evidence suggests that some of the observed
cellular senescence in cell culture may be due to inappropriate Primary cultures are generally subcultured at a 1 to 2 ratio
culture conditions as opposed to a predetermined replicative (they are split in half with each passage). Most continuous cell
higher split ratio. Passage number is generally the number of

senescence.2 Still other data support replicative senescence for lines replicate at higher rates and are subcultured at a much
the cells of some species (notably human) even when grown in
improved culture conditions. This senescence is mediated by times the cells have been subcultured into a new vessel. For
of population doublings (or population doubling level, PDL)

the shortening of the ends of the chromosomes (telomeres) diploid cultures, passage number is roughly equal to the number
with each cell division.3
since the culture was started. This is not the case for
In contrast, continuous (or immortalised) cell lines have infinite continuous cell lines as they are passaged at higher split ratios.
replicative capacity. These lines are derived from cell lines Consequently the PDL is not determined for continuous cell
through immortalisation or transformation by any one of a lines. In most cases, the PDL is an estimate as it does not
number of means. Many continuous cell lines were derived account for any cells that were lost due to death from necrosis
from tumor tissue. Most of the cell lines in the ATCC collection or apoptosis or cells which are nearing senescence and no
are continuous, though a few, such as CCD-1117Sk human skin longer divide.
fibroblast (ATCC® CRL-2465) or CCD-18Co human colon (ATCC®
CRL-1459™) are finite. For more information about ATCC
immortalised cell lines see the website.
As noted in the section on culture vessels, cell lines grow either

attached to a surface (anchorage dependent) or in suspension
(anchorage independent). As cells grow and divide in a
monolayer or in suspension, they usually follow a characteristic
growth pattern composed of four phases: Lag, log or exponential,
stationary or plateau and decline.
• Lag phase — Immediately after seeding of the culture

vessel, the cells grow slowly while recovering from the
stress of subculturing.
• Log or exponential phase — The cells enter a period of
exponential growth that lasts until the entire growth surface
is occupied or the cell concentration exceeds the capacity
of the medium.
• Stationary phase — Cell proliferation slows and stops.
• Decline phase — If the culture medium is not replaced and
the cell number is not reduced, the cells lose viability and Figure 1. Growth curve for cells grown in culture. Cells should be
their number decreases. subcultured while still in the exponential phase.
Order online at www.lgcstandards-atcc.org or contact your local LGC Standards office. See back cover for details. 7
Calculate the population doubling level with the following formula: 2. Monitor cell growth in the two media and watch for any
change in morphology or growth rate. If they are identical,
PDL = 3.32 (log Xe – log Xb) + S subculture the adapting cells at the next passage with a
1:2 split ratio in a 1:3 medium mix (25% original, 75% new).
Xb is the cell number at the beginning of the incubation time. 3. Monitor the growth rate and morphology of the original and
Xe is the cell number at the end of the incubation time. adapting cultures. At the next passage, split the adapting
S is the starting PDL. cultures 1:2 in a 1:7 medium mix (12.5% original, 87.5%
Calculate the population doubling time, or the time required for

new).
a culture to double in number, with the following formula:

4. Monitor the growth rate and morphology of the original and
adapting cultures. If the cells are identical, then at the next
passage split the adapting cells 1:2 in 100% new medium.
DT=T ln2/ln(Xe/Xb) At this point, the culture should be adapted to the new
medium.
T is the incubation time in any units.
Xb is the cell number at the beginning of the incubation time. To confirm complete adaptation to the new medium, perform
Xe is the cell number at the end of the incubation time. functional tests on cells derived from the original and new
medium. If at any point in the process the adapting culture fails
NOTE: Cells grow at different rates in each of the different to perform as well as the reference culture, then allow the
phases of the growth cycle and the calculated doubling time adapting culture more time and a few more passages in their
may be a composite of growth during more than one of these current medium mix (e.g., 1:3, 1:7, etc.) until they match the
phases. Growth during exponential growth or log phase is reference cells.
fairly constant and reproducible for a given set of growth
conditions. The same approach can be used to adapt cells to serum-free
medium; simply decrease the serum level in the medium by
ATCC tracks the PDL and passage number for many adherent half with each passage until a 0.06% (or lower) serum level is
cell lines when the depositor supplies this information at the time reached. At this point, the cells can be maintained in serum-free
of deposit. See the Product Information Sheet for the specific medium. If at any point the growth rate declines, then the serum
cell line for the passage number and/or PDL as part of the level should be increased to the level where the cells grew
batch-specific information supplied. normally. In this procedure, start with the “serum-free” medium
supplemented with serum so that only the level of serum
Adapting to a new medium or serum changes with each passage.
To ensure that the characteristics of your cell line remain Temperature

constant, maintain your cells in the same medium, serum, and
supplements with the same subculturing regimen used to Most animal cell lines require 37°C for optimum growth. Insect
establish the culture. Any change to the culturing conditions and amphibian cells require lower temperatures (such as 28°C)
has the potential to change the characteristics of the cell line. as do some animal cell lines which are temperature sensitive
for their phenotypic characteristics. While cultured cells can
Be particularly cautious when working with a new cell line as withstand considerable drops in temperature and most can
media formulations vary among suppliers, even for media with survive for several days at 4°C, few can tolerate even a few
similar or identical names. Read descriptions, formulations, and hours at more than 2°C above their optimal temperature.
labels carefully to ensure that the appropriate medium is used
or the cell line may be inadvertently adapted to a new medium. NOTE: Regularly calibrate the temperature control system of
All ATCC cell lines come with information on their growth incubators and use an alarm system when possible to warn
medium. In most cases, the recommended medium and serum against temperature increases above the optimum setting.
can be purchased from ATCC along with the cell line.
Examination of cultures
Use the following procedure to adapt a cell line to a new medium:
Observe the morphology and viability of cultures regularly and
1. Subculture the line at a 1:2 split ratio (split the culture in carefully. Examine the medium in the vessel for macroscopic
half) into two vessels. Maintain one with the original medium evidence of microbial contamination. This includes unusual pH
and continue to subculture these cells for the entire shifts (yellow or purple colour from the phenol red), turbidity, or
adaptation process. Use a 1:1 mix of the original and new particles. Also, look for small fungal colonies that float at the
medium in the second vessel. The culture grown in the medium-air interface. Specifically check around the edges of the
original medium serves as a reference point as well as a vessel as these may not be readily visible through the microscope.
safeguard in case the adapting cells do not survive the
process. The low split ratio helps mitigate the stress With an inverted microscope at low power (40x), check the
associated with subculturing as well as with the new medium. medium for evidence of microbial contamination and the
morphology of the cells. Bacterial contamination will appear as

small, shimmering black dots within the spaces between the
cells. Yeast contamination will appear as rounded or budding
particles, while fungi will have thin filamentous mycelia. For
non-adherent cells grown in flasks, such as hybridomas, this is
a simple matter of viewing the flask directly on the microscope.
For cells grown in spinner flasks or bioreactors, a sample of
the cell suspension will need to be withdrawn and loaded into a
microscope slide or hemocytometer for observation.
Most adherent cells should be attached firmly to the surface. In

some cases, healthy cells will round up and detach somewhat
during mitosis and appear very refractile. Following mitosis, they
will reattach. Some of these will float free if the culture vessel
is physically disturbed. In contrast, dead cells often round up
and detach from the monolayer and appear smaller and darker
(not refractile) than healthy cells.
Cells in suspension culture grow either as single cells or as

clusters of cells. Viable cells appear round and refractile whereas
dead cells appear smaller and darker. Occasionally, a portion of
the cells will attach and grow on the side of the culture vessel Figure 2. Hemocytometer grid with Neubauer ruling.
and appear round or flattened. The percentage of attached cells
varies with the culture conditions and the cell density. Cellular
debris may also be observed in healthy cell populations. Some
cell lines grow as mixed adherent and suspension cultures.
As a reference, photomicrographs for some ATCC cell lines are Count cells as follows:
available on the website. Cells are shown at two different
densities: just after subculturing (low) and just before they need 1. Clean, thoroughly dry, and assemble the hemocytometer
to be subcultured (high). with the cover slip.
2. Transfer a small amount of cell suspension to the edge of
In addition to daily examinations, periodically test a sample of each of the two counting chambers. Allow the cell suspension
the culture for the presence of fungi, bacteria, and mycoplasma. to be drawn into the counting chamber by capillary action.
There are several methods that can be used to check for these 3. Place the hemocytometer under an inverted microscope
contaminants. For additional information, refer to the section on and view the cells at 100x magnification.
microbial contamination (page 22). 4. Focus on the squares on each of four corners, labeled 1, 2,
3, and 4 in Figure 2.
Cell counting 5. Record the number of cells in each square. Average the
number of cells, and multiply by the dilution factor. If the
Cell counts are necessary in order to establish or monitor cells have not been diluted, this factor will be 104 cells/ml.
numbers. Hemocytometers (also spelled hemacytometers) are

growth rates as well as to set up new cultures with known cell Any dilution of the sample after it was removed from the
cell suspension, such as using vital stain, needs to be
commonly used to estimate cell number and determine cell included in the calculation.
viability. A hemocytometer is a fairly thick glass slide with two
counting chambers, one on each side. Each counting chamber For example, if the four counts are 60, 66, 69, and 75, the
has a mirrored surface with a 3 x 3 mm grid of 9 counting concentration would be 68 x 104 cells/ml for the sample
squares (see Figure 2). The chambers have raised sides that that was loaded into the hemocytometer. For best results,
will hold a coverslip exactly 0.1 mm above the chamber floor. adjust the concentration of the suspension so that 50 to
Each of the 9 counting squares holds a volume of 0.0001 ml. 100 cells are in each of the four counting squares.
Hemocytometers are excellent for determining cell viability, but Most cultures will grow at an initial inoculum cell concentration
are not precise for determining cell number due to the relatively ranging from 103 to 104 cells/cm2. Faster-growing cultures are
low number of cells actually counted. An automated counter usually set up at lower concentrations. Some cultures do not
will generate the most reliable data, particularly when used in grow well unless a minimum concentration of cells is initially
combination with the viability data from a hemocytometer. added; see the product sheet for details.
Cell viability and transferred into fresh culture vessels with the appropriate
growth medium where they will reattach, grow and divide.
Viability assays measure the number of viable cells in a
population. When combined with the total number of cells, the The procedure below is appropriate for most adherent cell lines.
number of viable cells provides an accurate indication of the However, since every cell line is unique, incubation times and
health of the cell culture. The most common and rapid methods temperature, number of washes or the solution formulations
rely upon the integrity of the cell membrane as an indicator of may vary. In all cases, continually observe the cells with a
cell viability. Both trypan blue and erythrosin B (ATCC® No. microscope during the dissociation process to prevent damage
30-2404) stains are actively excluded by viable cells but are by the dissociation solution. The amounts used in this procedure
taken up and retained by dead cells, which lack an intact are for a 75 cm2 flask. Adjust volumes as appropriate for
membrane. different sized vessels.
While both stains are used in the same way, ATCC recommends Monolayer subculturing
erythrosin B in place of trypan blue for hematopoetic cells. When
using trypan blue, incubate cells for two to five minutes prior 1. Bring the trypsin-EDTA solution (ATCC® No.30-2101),
to use. If not counted within this time, the cells will begin to balanced salt solution [Dulbecco’s Phosphate Buffered
deteriorate and take up the dye. Erythrosin B does not require Saline without calcium or magnesium, ATCC® No. 30-2200],
an incubation period. and complete growth medium to the appropriate temperature
for the cell line. In most cases, this is the temperature used
Erythrosin B stain generates more accurate results with fewer to grow the cells (usually 37°C). For some sensitive cells,
false negatives and false positives. Erythrosin B stain solution the trypsin-EDTA solution may need to be used at room
provides a clear background and does not bind serum proteins temperature or 4°C.
as avidly as trypan blue, making stained cells more distinct and 2. Remove and discard the cell culture medium from the flask.
easier to identify. Also, microbial contamination or precipitates 3. Rinse the cell monolayer with Dulbecco’s PBS without
in the cell culture are more readily apparent. Finally, trypan blue calcium or magnesium and remove.
is toxic and a potential carcinogen. 4. Add 2 to 3 ml of the trypsin-EDTA solution and incubate at
the appropriate temperature. Check the progress of cell
For either stain use the following directions: dissociation by microscopy. To avoid clumping, do not
agitate the cells by hitting or shaking the flask while waiting
1. Mix the cell suspension 1:1 with a 0.1% erythrosin B solution for them to detach.
in PBS or 0.4% trypan blue solution in PBS. 5. Once the cells appear to be detached (5 to 15 minutes for
2. Load the cells in the erythrosin B solution directly into a most cell lines; they will appear rounded and refractile under
clean, dry hemocytometer, but incubate the trypan blue the microscope), add 6 to 8 ml of complete growth medium
solution for two to five minutes before loading. with a pipette to the cell suspension to inactivate the trypsin.
3. Non-viable cells will be stained red (erythrosin B) or dark Gently wash any remaining cells from the growth surface of
blue (trypan blue). Cell viability is calculated as the number the flask. Check the cells with the microscope to be sure
of unstained or viable cells divided by the total number of that most (>95%) are single cells. If cell clusters are apparent,
cells and expressed as a percentage. continue to disperse the cells with gentle pipetting.
Subculturing monolayer cells NOTE: For serum-free or low-serum medium, remove the
at 125 x g) and then resuspend the cells in 6 to 8 ml of

trypsin-EDTA solution by gentle centrifugation (10 minutes
Anchorage-dependent cell lines growing in monolayers need to
be subcultured at regular intervals to maintain them in exponential fresh medium. In some cases, the trypsin will need to be
growth. When the cells are near the end of exponential growth inactivated with a trypsin inhibitor.
(roughly 70 to 90% confluent), they are ready to be subcultured.
The subculturing procedure, including recommended split-ratios 6. Add 12 to 15 ml of fresh culture medium to a new flask and
and medium replenishment (feeding) schedules, for each ATCC equilibrate this medium to the appropriate pH and temperature.
cell line is provided on the Product Information Sheet. 7. Count the cells in suspension and determine their viability
or simply divide them according to a routine split ratio and
Subcultivation of monolayers involves the breakage of both dispense them into the medium of the newly prepared flask.
intercellular and intracellular cell-to-surface bonds. For some cells Do not add a concentrated cell suspension to an empty
that are loosely attached, a sharp blow with the palm of your hand culture vessel as this can result in uneven cell attachment
against the side of the flask can dislodge them. Many require and growth.
the digestion of their protein attachment bonds with proteolytic 8. Place the flask back into the incubator. Examine the culture
enzymes such as trypsin/EDTA. For some cell lines mechanical the following day to ensure the cells have reattached and
forces such as scraping to dislodge the cells is preferred. After are actively growing. Change the medium as needed; for
the cells have been dissociated and dispersed into a single-cell most actively growing cultures two to three times per week
suspension, they are diluted to the appropriate concentration is typical.
Troubleshooting monolayer cell subculturing the dissociation agents is incorrect. Check these directly
Cells are difficult to remove.

and/or use a fresh bottle.
• The dispersed cell suspension was left too long at too high
a cell concentration prior to reseeding. Keep the cells on ice.
• Inhibitors in the medium (such as serum) have inactivated • The medium was faulty. Use the recommended formulation
the dissociating agents. Rinse the cell monolayer twice with and make sure it contains all of the required additives.
Dulbecco’s PBS without calcium or magnesium before
adding the dissociating solution. Or use the trypsin-EDTA Suspension cells
solution in place of the Dulbecco’s PBS for the first rinse
of the monolayer. Most primary cultures, finite cell lines, and continuous cell lines
• The dissociating solution was too weak. Use higher enzyme are anchorage dependent and thus grow in monolayers
concentrations, higher EDTA concentrations, or different attached to a surface. Other cells, particularly those derived
and/or additional enzymes (e.g., dispase, collagenase). from hematopoietic or certain tumor tissues, are anchorage
Or incubate the cells at 37°C to increase the activity of the independent and grow in suspension.
dissociating solution.
• The cells have been confluent for too long and the cell-to- Cell propagation in suspension has several advantages over
cell junctions are so tight they prevented the dissociation propagation in monolayer. Subculturing is a simple matter of
agents from reaching the substrate-cell interface. In the dilution. There is little or no growth lag after splitting a suspension
future, subculture the cells before they become as confluent. culture as there is with a monolayer culture, because there is
Cells form clumps after dissociation.

none of the trauma associated with proteolytic enzyme dispersal.
Suspension cultures require less lab space per cell yield, and
scale-up is straightforward. Cells can be propagated in
• The dissociation procedure was too harsh and genomic DNA bioreactors similar to the fermentors used for yeast or bacteria
was released from lysed cells. Either the pipetting was too cultures.
vigorous or the dissociating solution was too strong or too
toxic (i.e., the pH or osmolality of the buffer was incorrect). Depending upon the cell type, suspension cultures are seeded
Add a drop of sterile DNAse (1 mg/ml in water) to the cell at densities from 2 x 104 to 5 x 105 viable cells/ml and can
suspension to break down the DNA strands. In the future, attain densities of 2 x 106 cell/ml. If cells are seeded at too low
treat the cells more gently during pipetting, shorten the a density they will go through a lag phase of growth, grow very
incubation period, use a weaker dissociation solution slowly, or die out completely. If cell densities are allowed to
(lower the enzyme concentration or remove the EDTA), become too high, the cells may exhaust the nutrients in the
or incubate at a lower temperature. medium and die abruptly. Recommended seeding and
• The cells aggregated before dilution and dispersion into subculturing densities, media replenishment (feeding) schedules,
the medium. Hold the cell suspension on ice if there will be and medium formulations for each ATCC cell line are provided
a delay between removing the cells from the flask growth on the Product Information Sheet as well as in the catalogue
surface and seeding a new flask. description on the website.
• The cells were centrifuged too hard or too long when
gentle centrifugation (10 minutes at 125 x g).

removing excess dissociation solution. Be sure to use Suspension cell subculturing
Cells have difficulty reattaching to the flask.

1. Bring the complete growth medium to the appropriate
temperature (usually 37°C) in a water bath.
2. Thoroughly mix the cell/medium suspension; use a pipette
• The dissociation procedure was too long and stripped away to suspend cells grown in stationary flasks. Remove a
necessary attachment proteins from the cell membrane. small amount of the cell suspension to determine the cell
• Insufficient serum or attachment factors were present in the density and viability using a hemocytometer and vital stain
medium (common with serum-free medium). Add attachment (page 8).
factors to the medium and/or use a protein-coated flask 3. Calculate the volume of cells required to re-seed the flask
(collagen, poly-L-lysine, fibronectin, gelatin, etc.). at the minimum density for that cell line, taking into
• The dissociating solution was not inactivated or removed by consideration the amount of fresh medium that will be used.
centrifugation. Add additional serum or specific enzyme 4. Add the appropriate volume of medium to the culture vessel
medium or centrifuge (5 minutes at 125 x g) the cells down

inhibitors (e.g., soybean trypsin inhibitor) to the neutralising and then add the cell suspension. Do not add the
concentrated cell suspension to an empty flask. The same
from the dissociation solution and resuspend in fresh medium. culture vessel can be reused, but the chances of
Viability is lower than expected.

contamination increase with each reseeding due to the
buildup of small spills of medium on the flask opening.
5. If necessary, “gas“ the atmosphere of the flask with
• The dissociating procedure was too harsh. sterile-filtered CO2, seal the flask, and then incubate at the
• The pH or osmolality of the balanced salt solution containing appropriate temperature.
It is generally not necessary to completely change the medium suspension, they may need to be combined with cells from
unless the cells attain a very high density or the medium has an different flasks to achieve the necessary cell density.
acidic pH (yellow in colour from the phenol red). To completely 5. If there is a significant amount of cells attached to the walls
125 x g), decant the medium, and then resuspend the cells in
replace the medium, centrifuge the cells gently (10 minutes at of the culture vessel, particularly at the surface of the
medium, remove them with trypsin-EDTA and discard
fresh medium at the lower seeding density. them. If the cells in suspension are badly clumped, they
can be dispersed with the trypsin-EDTA solution, collected
Troubleshooting suspension cell subculturing by centrifugation, and then reseeded into the flask as the
Viability is lower than expected.

appropriate density. This treatment may be necessary for
the first few subcultures.
6. Continue to monitor the cells and subculture them every
• The cell suspension was left too long at too high a cell three days. Over time, they should adapt to growth in
concentration prior to subculture. In this case, the medium suspension and attain a constant growth rate.
will have a low pH and be yellow in colour. Completely
change the medium by gently centrifuging the cells and
resuspend in fresh medium at the lower seeding density.
• The cell suspension was diluted below the recommended
cell density range. Recover the cells by centrifugation and
resuspend in fresh medium at the appropriate cell density.
• The harvesting procedure was too harsh (pipetting too
vigorous, cells were centrifuged too hard or too long, cells
damaged during scraping or banging).
• The medium was faulty. Use the recommended formulation
and make sure it contains all of the required additives.
Adapting a monolayer cell line to grow in suspension
Some cell lines such as L-929 (ATCC® CCL-1™), HeLa (ATCC®

CCL-2™) and BHK-21 (ATCC® CCL-10™) can be adapted to
grow in suspension. With time, a population of cells can be
selected that does not self-aggregate or adhere to a growth
surface as readily as the parental line. However, the newly
selected line may have lost or acquired characteristics that are
different from the original cell population. In most cases it will be
necessary to maintain the culture in suspension with mechanical
stirring. Keep in mind that most anchorage-dependent cells will
grow in suspension only with the use of microcarrier beads.
The procedure below was developed for BHK-21 cells,4 but can
be used as a starting point for most cell lines.
1. Dissociate the cell monolayer using standard procedures.

Centrifuge and resuspend the cell suspension in an
appropriate spinner medium such as Joklik’s modified
Eagle’s Minimum Essential Medium (EMEM). Spinner media
have reduced levels of calcium and magnesium.
2. Count the cell suspension, and then seed two or more
spinner flasks with 5 x 105 cells/ml. This density may need
to be adjusted for your particular cell line. The sides of the
culture flask may need to be siliconised to prevent the cells
from sticking to the glass.
3. Observe the cultures daily. Remove samples and record
the number of viable cells for each flask.
centrifugation (10 minutes at 125 x g). Count, and re-seed

4. Every three days, collect the cells growing in suspension by
a fresh flask with fresh medium at 2.5 x 105 cells/ml.

Depending on how well (or not) the cells adapt to growth in
Complete growth media
A complete growth medium consists of a basal cell culture salt solution, non-essential amino acids, and sodium pyruvate.
medium supplemented with ingredients such as sera, growth It is formulated with a reduced sodium bicarbonate concentration
factors, trace elements, and hormones. There are numerous (1,500 mg/l) for use with 5% CO2 (see Sodium Bicarbonate and
formulations ranging from simple, basic mixtures containing the Buffering, page 13). Because EMEM is a simple medium, it is
minimum requirements for growing many cell lines to complex often fortified with additional supplements or higher levels of serum.
Dulbecco’s Modified Eagle’s Medium (DMEM) has roughly

serum-free mixtures specific for growing a single fastidious cell
line. The choice of a medium for a particular cell line is
somewhat empirical. twice the concentration of amino acids and four times the
amount of vitamins as EMEM, as well as ferric nitrate, sodium
Many medium formulations are available commercially in powder pyruvate, and some supplementary amino acids (though not all
or liquid form. non-essential amino acids). The original formulation contained
1,000 mg/l of glucose, but in the more commonly used variations
NOTE: Formulations can vary widely among suppliers, even for this amount was increased to 4,500 mg/l.
media with similar or identical names. Be sure to read catalogue
descriptions, formulations, and medium labels carefully to ensure ATCC DMEM (ATCC® No. 30-2002) has 4,500 mg/l of glucose
that the appropriate medium is used. For best results start cell and a reduced sodium bicarbonate concentration (1,500 mg/l)
cultures in the same medium used and distributed by ATCC for use with 5% CO2.
(listed on the Product Information Sheet).
Iscove’s Modified Dulbecco’s Medium (IMDM) was formulated for
ATCC lists complete medium formulations, plus all handling and growth of lymphocytes and hybridomas. Compared to DMEM, it
passage information, for all ATCC cell lines both in the online has additional amino acids, vitamins and inorganic salts.
catalogue description and on the Product Information Sheet Potassium nitrate was substituted for ferric nitrate. It also contains
that accompanies the cell line when shipped. Additionally, HEPES and selenium.
ATCC offers a full line of media, sera, and reagents for culturing
cells. These are the same reagents used at ATCC for cell growth ATCC IMDM (ATCC® No. 30-2005) has a reduced sodium
and propagation. Please contact us or refer to the website for bicarbonate concentration (1,500 mg/l) for use with 5% CO2.
details of ATCC cell culture products.
Hybri-Care Medium (ATCC® No. 46-X) is a combination and
Cell culture media modification of DMEM and NCTC 135 medium supplemented
with insulin, oxalacetic acid, and HEPES. It is based on the
Cell culture media are complex mixtures of salts, carbohydrates, formulation used by David H. Sachs and collaborators5 for the
vitamins, amino acids, metabolic precursors, growth factors, propagation of hybridomas and other fastidious cell lines.
hormones, and trace elements. The requirements for these
components vary among cell lines, and these differences are McCoy’s 5A and RPMI-1640 were developed at Roswell Park
partly responsible for the extensive number of medium Memorial Institute (RPMI) in Buffalo, New York. McCoy’s 5A
formulations. Carbohydrates are supplied primarily in the form of (ATCC® No. 30- 2007) was originally used to grow Novikoff
glucose. In some instances, glucose is replaced with galactose hepatoma cells and will support the growth of primary cultures.
to decrease lactic acid build-up, as galactose is metabolised at
a slower rate. Other carbon sources include amino acids RPMI-1640 is a modification of McCoy’s 5A and was
(particularly L-glutamine) and pyruvate. developed for the long-term culture of peripheral blood
lymphocytes. RPMI-1640 will support the growth of a wide
In addition to nutrients, the medium helps maintain the pH and variety of cells in suspension as well as a number of cells
osmolality in a culture system. The pH is maintained by one or grown as monolayers.
more buffering systems; CO2/sodium bicarbonate, phosphate,
and HEPES are the most common. Sera will also buffer a ATCC RPMI-1640 (ATCC® No. 30-2001) was modified to contain
complete medium. Phenol red, a pH indicator, is added to higher amounts of glucose (4,500 mg/l), sodium pyruvate, and
medium to colourimetrically monitor changes in pH. HEPES buffer. It also contains a reduced concentration of
sodium bicarbonate (1,500 mg/l) for use with 5% CO2.
Commonly used culture media include the following:
Ham’s Nutrient Mixtures were originally developed to support
Eagle’s Minimum Essential Medium (EMEM) was among the the clonal outgrowth of Chinese hamster ovary (CHO) cells
first widely used media and was formulated by Harry Eagle (ATCC® CCL-61™). As with EMEM, there have been numerous
from his earlier and simpler basal medium (BME). BME was modifications to the original formulation including Ham’s F-12
developed for culturing mouse L cells (ATCC® CCL-1™) and HeLa medium, a more complex formulation than the original F-10
cells (ATCC® CCL-2™). Over time, there have been numerous suitable for serum-free propagation.
variations on the EMEM formula for different applications.
Kaighn’s modification of Ham’s F-12 (Ham’s F-12K) was
ATCC EMEM (ATCC® No. 30-2003) contains Earle’s balanced designed to support the growth and differentiation of primary
cells with or without serum. F-12K has increased amounts of there is free exchange of the atmosphere immediately above
amino acids, pyruvate, biotin, calcium, magnesium, putrescine, the medium with the atmosphere of the incubator) or in closed
and phenol red in addition to other modifications from the F-12 systems (where the two atmospheres are kept separate). The
formula. buffering system employed in the medium needs to be matched
to the culture system. Otherwise the cells may be subject to
ATCC Ham’s F-12K (ATCC® No. 30-2004) has a reduced sodium metabolic stress which will impair their performance.
bicarbonate concentration (1,500 mg/l) for use with 5% CO2
DMEM/F12 Medium is a 1:1 mixture of Dulbecco’s modified

In closed systems the level of CO2 is regulated by the metabolism
of the cells. The culture vessel must be sealed (flasks tightly
EMEM and Ham’s F-12. It is an extremely rich and complex capped) to retain any CO2 generated by the cells. Consequently,
medium and will support the growth of a broad range of cell closed systems provide additional protection against
types in both serum and serum-free formulations. contamination and have simpler incubator requirements than
open systems. Closed systems usually require media with
ATCC DMEM/F12 medium (ATCC® No. 30-2006) has a reduced buffers based on Hanks’ balanced salt solution having relatively
sodium bicarbonate concentration (1,500 mg/l) for use with low levels of sodium bicarbonate.
5% CO2.
Leibovitz’s L-15 Medium (ATCC® No. 30-2008) is formulated for

In open systems, humidity (to reduce evaporation) and a
means of regulating CO2 levels (if the culture medium contains
use without CO2 incubation as is found in teaching laboratories sodium bicarbonate) are required during incubation to maintain
or when collecting biopsy samples. The standard sodium the pH of the culture medium. Open systems usually require
bicarbonate/CO2 buffering system is replaced by a combination the higher levels of sodium bicarbonate found in Earle’s salt
of phosphate buffers, free-base amino acids, higher levels of solution combined with a 5 to 10% CO2 atmosphere supplied
sodium pyruvate, and galactose. Cell cultures can be grown in by the incubator. In general, 1.2 to 2.2 g/l of sodium bicarbonate
CO2 incubators with L-15 medium provided there is no is used with 5% CO2 whereas 3.7 g/l sodium bicarbonate is
exchange between the air in the culture vessel with that of the used with 10% CO2. The exact amount will depend upon the
incubator (i.e., caps of flasks are tightly closed). medium formulation.
Media formulations In some cases, researchers “gas” the atmosphere of the

culture vessel with a stream of sterile-filtered 5% CO2/95% air
Formulations of media available from ATCC can be found online. mixture and then tightly seal the flask prior to incubation in a
There are cell lines in the collection that require media not nonhumidified and non-CO2 incubator.7 While these culture
currently sold by ATCC. Some of these media formulations vessels work with simpler non-humidified, non-CO2 incubators,
have been provided on page 27. the medium requirements are those of an open system.
Media ingredients All ATCC media, with the exception of Leibovitz’s L-15 (ATCC®
Sodium bicarbonate and buffering

No. 30-2008), are designed to be used with 5% CO2 levels.
Most have a sodium bicarbonate concentration of 1.5 g/l and
are supplemented with extra sodium pyruvate. ATCC
Cells produce and require small amounts of carbon dioxide modification of McCoy’s 5A (ATCC® No. 30-2007) has a slightly
for growth and survival.6 In culture media, dissolved CO2 is in higher level of sodium bicarbonate (2.2 g/l) and does not
equilibrium with bicarbonate ions and many medium contain sodium pyruvate.
formulations take advantage of this CO2/bicarbonate reaction
to buffer the pH of the medium. CO2 dissolves freely into the While most commercial formulations of liquid media do contain
medium and reacts with water to form carbonic acid. As the the appropriate amount of sodium bicarbonate, it is generally
cells metabolise and produce more CO2, the pH of the medium omitted from the powdered form and needs to be added
decreases as the chemical reaction below is driven to the right: before use.
H2O + CO2 H2CO3 H+ + HCO3- Some medium formulations incorporate other buffering systems
such as phosphate or HEPES in addition to CO2/sodium
The optimal pH range of 7.2 to 7.4 can be maintained by bicarbonate. These media have the advantage of maintaining
supplementing the medium with sodium bicarbonate and optimal pH in an open system when the culture vessel is
regulating the level of CO2 in the atmosphere above the removed from the enriched CO2 atmosphere of the incubator.
medium as shown by the reaction below:
HEPES buffer
H2O + CO2 + NaHCO3 H+ + Na+ + 2HCO3-
HEPES and other organic buffers can be used with many cell
In tissue culture, cells are grown either in open systems (where lines to effectively buffer the pH of the medium.8 Indeed, some
standard medium formulations include HEPES. However, this Use caution when adding more L-glutamine than is called for in
compound can be toxic, especially for some differentiated cell the original medium formulation. L-Glutamine degradation results
types, so evaluate its effects before use.9 HEPES has been in the build-up of ammonia which can have a deleterious effect
shown to greatly increase the sensitivity of media to the on some cell lines. For most cell lines, ammonia toxicity is more
phototoxic effects induced by exposure to fluorescent light.10,11 critical for cell viability than L-glutamine limitation.
Phenol red Non-essential amino acids
Phenol red is used to monitor the pH of media. During cell All medium formulations contain the ten essential amino acids
growth, the medium changes colour as it changes pH due to as well as cysteine, glutamine, and tyrosine. The inclusion of
metabolites released by the cells. At low pH levels, phenol red the other non-essential amino acids (alanine, asparagine,
turns the medium yellow, while at higher pH levels it turns the aspartic acid, glycine, glutamic acid, proline, and serine) in
medium purple. For most tissue culture work (pH 7.4), the some medium formulations reduces the metabolic burden on
medium should be bright red. the cells allowing for an increase in cellular proliferation.
Unfortunately, phenol red can mimic the action of some steroid Sodium pyruvate
hormones, particularly oestrogen. For studies with oestrogen- Pyruvate is an intermediary organic acid metabolite in glycolysis
sensitive cells, such as mammary tissue, use media without and the first component of the Embden-Meyerhof pathway. It
phenol red. Additionally, the sodium-potassium ion homeostasis can pass readily into or out of the cell. Its addition to tissue
is upset when phenol red is included in some serum-free culture medium provides both an energy source and a carbon
formulations; this effect is neutralised by the inclusion of serum skeleton for anabolic processes. Pyruvate may help in
or bovine pituitary hormone in the medium.12 Phenol red is maintaining certain specialised cells, in clonal selection, in
frequently omitted from studies with flow cytometry as its colour reducing the serum concentration of the medium,7 and in
interferes with detection. reducing fluorescent light-induced phototoxicity.10 Cellular
L-Glutamine
metabolism of pyruvate produces carbon dioxide which is given
off into the atmosphere and becomes bicarbonate in the medium.
Sodium pyruvate is added to give a final concentration of
L-Glutamine (ATCC® No. 30-2214) is an essential amino acid 1 mM in most media, but is increased to 5 mM in Leibovitz’s
required by virtually all mammalian and insect cells grown in L-15 medium primarily to facilitate use in CO2-free environments.
culture. It is used for protein production, as an energy source,
and in nucleic acid metabolism. It is also more labile in liquid Media supplements
cell culture media than other amino acids. The rate and extent
of L-glutamine degradation are related to storage The complete growth media recommended for some cell lines
temperatures, age of the product, and pH. requires the addition of components not already available in the
base media and serum. These components include hormones,
Because L-glutamine is so labile, it is often omitted from growth factors and signaling substances that sustain proliferation
commercial liquid medium preparations to lengthen the product and maintain normal cell metabolism.
shelf life. In these cases, it must be aseptically added prior to
use. L-Glutamine is not as labile in dry form and most Supplements are usually prepared as 100x (or higher) stock
powdered medium formulations do include it. solutions in serum-free medium. Some supplements may need
to be dissolved in a solvent prior to subsequent dilution in serum-
In some cases, the addition of L-glutamine to complete cell free medium to the stock concentration. Stock concentrations
culture medium can extend the usable life of the medium. If should be aliquoted into small volumes and stored at an
L-glutamine is suspected to be a limiting factor during cell culture, appropriate temperature; most stock concentrations can be
a simple test of ‘spiking’ the medium with a small amount of stored at –80ºC, but check with your supplier prior to storing.
L-glutamine will determine whether or not more is required.
Simply add a small amount of L-glutamine (~2 mM final The addition of supplements can change the final osmolality of
concentration) to the culture medium. If the cell growth rate the complete growth medium, which may have a negative effect
increases, L-glutamine is most likely deficient and more should on the growth of cells in culture. It is best to recheck the
be added. Alternately, the concentration of L-glutamine can be osmolality of the complete growth medium after small volumes
measured directly by standard analytical means such as HPLC of supplement stock solutions are added; optimal osmolality for
(High Performance Liquid Chromatography). most vertebrate cell lines should fall between 260 and 320
mOSM/kg.
L-Glutamine concentrations for mammalian cell culture media
can vary from 0.68 mM in Medium 199 to 4 mM in Dulbecco’s After supplements have been added to a base medium, the
Modified Eagle’s Medium. Invertebrate cell culture media, such shelf life of the complete growth medium should be determined
as Schneider’s Drosophila medium, may contain as much as on a case-by-case basis. Complete media containing protein
12.3 mM L-glutamine. supplements (e.g., epidermal growth factor, bovine serum
albumin, etc.) tend to degrade faster than base media alone. serve as a valuable prophylactic. For example, antibiotic use is
Most complete growth media can be stored in aliquots at 2 to recommended when developing and working with primary culture
8ºC for about a month. However, if any supplement is expected and when using flow cytometry to isolate subpopulations.
to expire before the one-month period has passed, the expiration
date for the complete growth media should follow suit. Some Animal sera
fastidious cell lines may require that components be added
immediately before use. Do not freeze complete growth medium. Sera serve as a source for amino acids, proteins, vitamins
Freezing cell culture media at -70ºC or below causes some of (particularly fat-soluble vitamins such as A, D, E, and K),
the growth factors and/or vitamins to precipitate out of solution. carbohydrates, lipids, hormones, growth factors, minerals, and
It can be very difficult to get these components to go back into trace elements. Additionally, serum buffers the culture medium,
solution after thawing, even if warmed to 37ºC. ATCC inactivates proteolytic enzymes, increases medium viscosity
recommends storing media between 2 and 8ºC, away from light. (which reduces shear stress during pipetting or stirring), and
conditions the growth surface of the culture vessel. The exact
For additional information regarding the preparation, storage, or composition is unknown and varies from lot to lot, although
usage of specific supplements, contact your local supplier or lot-to-lot consistency has improved in recent years.
consult with the manufacturer’s Product Information Sheet.
Sera from fetal and calf bovine sources are commonly used
Osmolality to support the growth of cells in culture. Fetal serum is a rich
source of growth factors and is appropriate for cell cloning and
The osmolality of cell culture media for most vertebrate cells is for the growth of fastidious cells. Calf serum, because of its
kept within a narrow range from 260 to 320 mOsm/kg, even lower growth-promoting properties, is used in contact-inhibition
though most established cell lines will tolerate a rather large studies with NIH/3T3 cells (ATCC® CRL-1658™). In contrast to
variation in osmotic pressure. In contrast, the osmolality fetal or calf sera, horse serum is collected from a closed herd
requirements for some invertebrate cell lines fall outside of this of adult animals ensuring lot-to-lot consistency. Horse serum is
range. For example, the snail embryo (ATCC® CRL-1494™) less likely to carry the contaminants found in bovine sera such
requires medium of about 155 mOsm/kg, while some insect as viruses and less likely to metabolise polyamines which may
cells prefer 360 to 375 mOsm/kg. Most commercially available be mitogenic for some cells. Horse and bovine calf sera are
liquid media report osmolality and it is advisable to check the less expensive and more readily available than fetal bovine
osmolality of any medium after the addition of saline solutions, serum. The pricing and availability of fetal serum fluctuates
drugs or hormones dissolved in an acid or base solution, or considerably.
large volumes of buffers (e.g., HEPES).
Unfortunately, naturally derived products from bovine sources
Antibiotics and antimycotics may contain adventitious viruses such as bovine viral diarrhoea
virus (BVDV), bovine parvovirus, bovine adenovirus, and blue
Antibiotics and/or antimycotic agents are added to cell culture tongue virus. All reputable suppliers test their products for
media as a prophylactic to prevent contamination, as a cure infectious virus by several methods including fluorescent
once contamination is found, to induce the expression of antibody, cytopathic effect, and hemadsorption. These products
recombinant proteins, or to maintain selective pressure on are also screened for the standard microbial contaminants such
transfected cells. as bacteria, fungi, and mycoplasma.
Routine use of antibiotics or antimycotics for cell culture is not BVDV, in contrast to the other virus contaminants, is present in
recommended unless they are specifically required, such as nearly all bovine serum at very low levels even when tests for
G418 for maintaining selective pressure on transfected cells. infectious virus are negative. Fortunately, very few cell lines
Antibiotics can mask contamination by mycoplasma and resistant (except those of bovine origin) are susceptible to this virus. For
bacteria. Further, they can interfere with the metabolism of the few sensitive cell lines, use non-bovine sera or irradiated
sensitive cells. Avoid antimycotics as they can be toxic to many bovine sera. Several ATCC cell lines were tested for BVDV
cell lines. contamination14 and the results of this study are indicated in
the cell line description on the website. Bovine-derived products
While cell lines can be cured of microbial contamination with also may contain the agent responsible for bovine spongiform
antibiotics and/or antimycotics, this is not recommend unless encephalopathy (BSE). Unfortunately, there is no test for the
the cell line is irreplaceable; the process is lengthy and there is presence of this agent and we highly recommend that you obtain
no guarantee contamination will be eliminated. Even if the all bovine products (including sera) from countries not affected
contamination is eliminated, there is no way of ensuring that by BSE such as the United States, Australia and New Zealand.
the resulting cell line will have the same characteristics as the
initial one due to the stress of the treatment. It is best to discard At one time animal serum was a major source of mycoplasma
the cell line and start over with new stocks. Mycoplasma contamination of tissue culture cells. However, nearly all sera
contamination in particular is very difficult to eliminate (see page today are filtered through several 0.1 m pore (or smaller) filters
22). In some cases, antibiotic use for short periods of time can which effectively remove this organism.
ATCC offers the following four types of animal sera: Heat inactivation was originally performed to inactivate
complement (a group of proteins present in sera that are part
• Fetal Bovine Serum (also known as fetal calf)
of the immune response) as well as to destroy mycoplasma
ATCC® No. 30-2020
contaminants. Today, mycoplasma contamination, if any, is
• Fetal Bovine Serum qualified for embryonic stem cells
removed by filtration. Removal of complement is usually
ATCC® No. SCRR-30-2020
unnecessary, but can be important when preparing or assaying
• Iron-supplemented Calf Bovine Serum
viruses or in cytotoxicity tests. According to a study by HyClone,15
ATCC® No. 30-2030
warming serum to 37°C inactivates heat-labile complement
• Horse Serum
factors. A few types of cell lines grow better in heat-inactivated
ATCC® No. 30-2040
sera such as embryonic stem cells16 and many insect cell lines.17
These products are rigorously tested for adventitious infective
The following procedure can be used to heat-inactivate serum:
agents and sourced from only U.S. herds. Further, each lot is
tested for its ability to support cell growth and is the same sera
1. Thaw serum.
used in ATCC labs.
2. Preheat a water bath to 56°C. Use sufficient water to
Storage
immerse the bottle above the level of serum.
3. Mix thawed serum by gentle inversion and place in the 56°C
bath. The temperature of the water bath will drop.
Store sera at -20°C or colder for storage over 30 days. ATCC
4. When the temperature of the water bath reaches 56°C
sera are routinely stored at -70°C. Do not store sera at
again, continue to heat for an additional 30 minutes. Mix
temperatures above -20°C for any length of time. Avoid repeated
gently every 5 minutes to insure uniform heating.
freeze-thaws by dispensing and storing in aliquots.
5. Remove serum from water bath, cool quickly (slow cooling
Thawing
can sometimes reverse the inactivation of complement
activity), and store at -20°C or colder.
The following procedure is used to thaw serum:
1. Place frozen serum in a refrigerator at 2 to 8°C overnight.

2. Put the bottles in a 37°C water bath and gently agitate from
time to time to mix the solutes that tend to concentrate at the
bottom of the bottle.
Do not keep the serum at 37°C any longer than necessary to

thaw it, and do not thaw the serum at higher temperatures.
Thawing serum in a bath above 40°C without mixing may lead
to the formation of a precipitate inside the bottle.
Turbidity and precipitates
All sera may retain some fibrinogen. Because external factors

may initiate the conversion of fibrinogen to fibrin, flocculent
material or turbidity may be observed after serum is thawed.
The presence of this material does not alter the serum’s
performance. If the presence of flocculent material or turbidity
is a concern, it can be removed by filtration through a 0.45 µm
filter. A precipitate can form in serum when incubated at 37°C
or higher for prolonged periods of time which may be mistaken
for microbial contamination. This precipitate may include crystals
of calcium phosphate, but does not alter the performance of the
serum as a supplement for cell culture. Heat inactivation of sera
can also cause the formation of precipitates.
Heat inactivation
ATCC does not routinely use heat-inactivated serum unless

specifically required for a particular cell line. Heat inactivation
is usually unnecessary and can be detrimental to the growth
of some cells. It will reduce or destroy growth factors present
in the serum.
Culture vessels and surfaces
Vessels Next, decide whether the cells will be grown as an open system
or as a closed system (see the section on sodium bicarbonate,
Culture vessels provide a contamination barrier to protect the page 13). Open-system plastic dishes are less expensive than
cultures from the external environment while maintaining the closed-system flasks, but require more expensive incubators that
proper internal environment. For anchorage-dependent cells, can regulate the CO2 and humidity in the atmosphere. Closed
the vessels provide a suitable and consistent substrate for cell systems provide additional protection against contamination
attachment. Other characteristics of vessels include easy and have simpler incubator requirements.
access to the cultures and optically clear viewing surfaces.18
All dishes and multiwell plates are open systems. All other culture
Originally all culture vessels were glass. Drawbacks for glass vessels can be used in either mode by leaving caps loose for
include the heavy weight, expense, labor-intensive cleaning, and an open system or tightened for a closed system. The plastic
poor microscopic viewing compared to plastic. By the 1960s, walls of culture vessels are slightly permeable to carbon dioxide
surface treatment techniques were developed for polystyrene, and oxygen, permitting a very small amount of gas exchange.
allowing plastic vessels to replace glass for most cell culture This is not a problem in most culture applications, but may
applications. interfere with anoxia experiments or long-term storage of
media.19 Caps that allow gas exchange when the cap is fully
The information below focuses on standard culture vessels used by tightened are available to reduce opportunities for flask spills
many researchers. Large-scale culture equipment is not included. and contamination in open systems.
Selecting the right vessel The last step is matching the desired cell yield with an
appropriately sized culture vessel. For monolayer cultures, the
First, match the characteristics of the cells to be grown with the yield is limited by the area of treated growth surface.
characteristics of the different culturing systems. There are Approximately 0.5 x 105 to 1 x 105 cells/cm2 of treated surface
three basic types of cell cultures: is a typical yield for confluent continuous mammalian cell lines.
For suspension cultures the total cell yield is determined by the
• Anchorage dependent, which must become attached to a working volume of the vessel. In stirred systems, cell
surface to grow (for example, human diploid fibroblasts). concentrations can easily reach between 1 x 106 and 2 x 106
• Anchorage independent, which grow in suspension (most cells/ml of medium. However, the exact yields will need to be
blood-derived cell cultures). determined empirically for each cell line. ATCC strongly
• Cells that can grow either attached or in suspension (many recommends that cells be maintained in the logarithmic phase
transformed cell lines such as HeLa and BHK-21). of growth, and not be allowed to enter the stationary phase.
Anchorage-dependent cell lines are routinely passaged or split
Understand the growth requirements of the cultures to help select before they reach confluency.
Flasks
the best culture system. There are four basic culture systems:
• Stationary monolayer cultures which are grown in undisturbed

flasks, dishes, and multiwell plates. These are the easiest Alexis Carrel developed the first glass flasks in the 1920s. Harry
culture systems to use and require the least amount of Earle developed the more traditional straight neck rectangular
equipment. However, these systems are very labour (also hexagonal) glass T-flasks in the 1940s. Today, plastic flasks
intensive for producing large quantities of cells. are available with a range of growing areas, a variety of
• Moving monolayer cultures which are grown primarily in roller shapes, with several different neck designs. Choice of design
bottles. These vessels are slowly rotated (approximately depends on the cell culture techniques used as well as personal
0.5 to 1 rpm) on motorised racks or drums and are widely preference. The more common sizes are listed below.
used for producing large quantities of cells. Roller bottles
employ simple technology but require an investment in the Description Growth Recommended working Cell yield*
appropriate equipment. area (cm2) volume (ml)
• Stationary suspension cultures which are grown without T-25 25 5 to 10 2.5 x 106
agitation in untreated dishes and flasks. These are best for T-75 75 15 to 25 7.5 x 106
growing small volumes of anchorage-independent cells that T-150 150 30 to 50 15.0 x 106
T-175 175 35 to 60 17.5 x 106
grow poorly in traditional stirred suspension cultures.
T-225 225 45 to 75 22.5 x 106
• Moving suspension cultures which are grown in mechanically
*Cell line dependent. Based upon a density of 1 x 105 cells/cm2.
stirred vessels (spinner flasks), bioreactors, or fermentors.
These systems are the most economical in terms of space,
labour and media; as a result, stirred suspension cultures
are usually the method of choice for producing large volumes
of cells both in the lab and in industry. Many anchorage-
dependent cells can be adapted to grow on microcarriers
to take advantage of these systems.
Culture vessels and surfaces
Cell culture dishes Surface coatings and feeder cells
Cell culture dishes offer the best economy and access to the Most tissue culture work uses disposable polystyrene vessels.
growth surface. This makes them the vessels of choice for The vessel surface is treated to render it hydrophilic (wettable).
cloning or other manipulations such as scraping that require Most cell lines in the ATCC collection are cultivated on treated
direct access to the cell monolayer. They must be used with plastic surfaces in dishes, flasks, or roller bottles. Since the
incubators that control CO2 and humidity. Most manufacturers properties of tissue culture plastic can vary among
offer dishes in four diameters: 35 mm, 60 mm, 100 mm, and manufacturers, samples should be evaluated for their ability to
150 mm. These are nominal diameters and may not be the support cell growth and propagation prior to use. ATCC
actual diameter of the growth surface. Cell culture dishes are routinely uses the SelecT™ fully automated cell culture system.
available with either specially treated surfaces for growing
anchorage-dependent cells, or untreated (native) surfaces for Some fastidious cell lines require further treatment of the
growing suspension cultures where attachment is not desired. growth surface before they will attach and proliferate. The most
common techniques include coating the surface with serum,
Diameter (mm) Growth area (cm2) Working volume (ml) Cell yield* collagen (ATCC® No. 30-2511), laminin (ATCC® No. 30-2505),
35 8 1 to 2 0.8 x 106 gelatin, poly-L-lysine, or fibronectin.
60 21 4 to 5 2.1 x 106
100 55 10 to 12 5.5 x 106 Beyond simple attachment, some cells require specialised
150 148 28 to 32 14.8 x 106 surface treatment in order for them to differentiate into more
tissue-like formations. For example, endothelial cells will form
Multiwell plates tubules and neuronal cells will extend neurite processes when
cultured on a surface of extracellular matrix (ECM) proteins.
These widely used vessels were originally designed for virus These ECM proteins closely resemble the basal lamina
titration, but have since become popular in many other membrane surrounding cells in tissue and not only provide
applications, especially hybridoma production, high-throughput attachment points, but modulate signal transduction from
screening, and toxicity testing. Multiwell plates offer significant external growth factors and hormones, influence the
savings in space, media, and reagents when compared to an permeability of ions and nutrients, and actively “communicate”
equal number of dishes. They are more convenient to handle, with intracellular processes through integrins.
especially if the pipettors, plate washers, readers, and other
equipment for processing these plates are used. They must Finally, some cells, particularly when seeded at low densities
be used with incubators that control humidity and CO2 levels. as for cloning, require the support of living cells. Most cells are
“happier in a crowd.” Feeder layer cells supply a crowd by
Description Growth area/ Working volume/ Cell yield*
conditioning the medium through metabolic leakage and/or the
well (cm2) well (ml) active secretion of growth and other factors. They also provide
96-well 0.32 0.1 to 0.2 0.32 x 105 a support matrix for cell attachment and proliferation. To prevent
48-well 1.0 0.3 to 0.6 0.8 x 105 feeder layer cells from overgrowing the cells of interest, they are
24-well 1.88 0.5 to 1.2 1.9 x 105 treated to prevent division. Common methods include irradiation
12-well 3.83 1.0 to 2.4 3.8 x 105 with X-rays or gamma rays or treatment with mitomycin C. Each
6-well 9.40 2.0 to 3.0 9.5 x 105 of these treatments damages cellular DNA so that the cells
*Cell line dependent. Based upon a density of 1 x 105 cells/cm2. continue to metabolise but can no longer proliferate. ATCC
Roller bottles offers a variety of well-characterised feeder cells. Contact us for
more details.
The roller bottle was developed for cultivating large numbers
of anchorage-dependent cells.20 Today they provide a more
economical means for cultivating large volumes of cells using
essentially the same culture techniques as with flasks but with
considerably less labour. Besides the traditional smooth wall
design, roller bottles are available with small ridges that
approximately double the surface area available for growing
cells without increasing the dimensions of the bottles.
Description Growth area (cm2) Working volume (ml) Cell yield*

Small 490 100 to 150 4.9 x 107
Standard 850 170 to 250 8.5 x 107
Pharmaceutical 1750 340 to 500 17.5 x 107
Cryopreservation
Most cell cultures can be stored for many years, if not indefinitely, Freeze medium
at temperatures below -130°C (cryopreservation). ATCC has
recovered cells from cultures cryopreserved for more than 40 Glycerol and DMSO at 5 to 10% are the most common
years. The many advantages of cryopreservation far outweigh cryoprotectant agents. While DMSO can be toxic to cells, it
the required investment in equipment and reagents. These penetrates them much faster than glycerol and yields more
advantages include: reproducible results. Unfortunately, DMSO can cause some
cells to differentiate (e.g., HL-60 promyeloblast cells) and may
• Generation of safety stocks to ensure against loss of the be too toxic for some cells (e.g., HBE4-E6/E7 lung epithelial
culture from equipment failures or contamination by cells). Glycerol should be used in these instances. Glycerol can
microorganisms or other cell lines. be sterilised by autoclaving whereas DMSO must be sterilised
• Elimination of the time, energy, and materials required to by filtration. Care should be used when handling any DMSO
maintain cultures not in immediate use. solution as it will rapidly penetrate intact skin and may carry
• Preservation of cells with finite population doublings (that toxic contaminants along with it.
will ultimately senesce).
• Insurance against phenotypic drift in the culture due to Use only reagent grade (or better, such as cell culture grade)
genetic instability and/or selective pressure. DMSO or glycerol. Store both in aliquots protected from light.
• Creating a standard reagent to be used for a series of ATCC offers DMSO (ATCC® No. 4-X) that has been thoroughly
experiments. tested for cell culture use.
Overview For cells grown in serum-free medium, adding 50% conditioned

medium (serum-free medium in which the cells were grown for
As the cell suspension is cooled below the freezing point, ice 24 hours) to both the cell freezing and the recovery medium
crystals form and the concentration of the solutes in the may improve recovery and survival. The addition of 10 to 20%
suspension increases. Intracellular ice can be minimised if cell culture grade bovine serum albumin to serum-free freezing
water within the cell is allowed to escape by osmosis during the medium may also increase post-freeze survival.
cooling process. A slow cooling rate, generally -1°C per minute,
facilitates this process. However, as the cells lose water, they Other variations of freeze medium formulations include high
shrink in size and will quickly lose viability if they go beyond a (up to 90%) concentrations of serum which presumably supplies
minimum volume. The addition of cryoprotectant agents such as some cryoprotection as well as additional growth factors; use of
glycerol or dimethylsulfoxide (DMSO) will mitigate these effects. a balanced salt solution designed for hypothermal conditions in
place of medium designed for 37°C incubation; and the addition
The standard procedure for cryopreservation is to freeze cells of apoptotic inhibitors which may prevent delayed onset cell
slowly until they reach a temperature below -70°C in medium death following recovery.21 Optimum formulations for individual
that includes a cryoprotectant. Vials are transferred to a liquid- cell lines need to be determined empirically.
nitrogen freezer to maintain them at temperatures below -130°C.
Equipment
Cryopreservation vials
The recovery of cryopreserved cells is straightforward: Cells are
thawed rapidly in a water bath at 37°C, removed from the
freeze medium by gentle centrifugation and/or diluted with
growth medium, and seeded in a culture vessel in complete There are two materials to choose from for cryopreservation
growth medium. vials: glass or plastic. Glass vials are more difficult to work
with; they need to be sterilised before use, they do not come
There are numerous factors which affect the viability of recovered with labels (information is imprinted into the glass), they need
cells. Modify the procedure for each cell line to attain optimal to be sealed with a hot flame, and they can be difficult to open.
cell viability upon recovery. Some of the critical parameters for However, they are preferred for long-term storage (many years)
optimisation include the composition of the freeze medium, the of valuable cultures and are considered fail-safe once properly
growth phase of the culture, the stage of the cell in the cell sealed. ATCC uses glass vials for the storage of seed stocks
cycle, and the number and concentration of cells within the which are placed in the lower level of the liquid nitrogen tank.
freezing solution.
Plastic vials are used for the storage of distribution stocks.
ATCC provides information on cryopreservation for all cell lines Plastic vials come in two varieties: those with an internal thread
on the Product Information Sheet. Most ATCC cell lines are and silicone gasket and those with an external thread. The
frozen with a cryopreservation medium consisting of 5% internal thread version was the first commercially available, but
DMSO and complete growth medium. has some disadvantages over the external thread version. For
example, while the silicone gasket provides an excellent seal,
it needs to be tightened just right; too tight or too loose and the
vial will leak.
Cryopreservation
Controlled-rate freezing chambers 3. Collect cells by gentle centrifugation (10 minutes at 125 x g)
and resuspend them in the freeze medium at a concentration
There are several means to achieve a cooling rate of -1°C per of 1 x 106 to 5 x 106 viable cells/ml. Continue to maintain
minute. The best is with a computer controlled, programmable the cells in culture until the viability of the recovered cells
electronic freezing unit (such as CryoMed Freeze) which is confirmed (see Step 9).
rigorously maintains this rate of cooling. This is the method used 4. Label the appropriate number of vials with the name of the
exclusively at ATCC. Such equipment is relatively expensive cell line and the date. Then add 1 to 1.8 ml of the cell
and absolutely necessary for only the most sensitive cells. suspension to each of the vials (depending upon the
volume of the vial) and seal.
A less costly approach is to place the cryopreservation vials into 5. Allow cells to equilibrate in the freeze medium at room
an insulated chamber and cool for 24 hours in a mechanical temperature for a minimum of 15 minutes but no longer
freezer at -70°C or lower. There are several commercially than 40. This time is usually taken up in dispensing aliquots
available freezing chambers which achieve a cooling rate very of the cell suspension into the vials. After 40 minutes, cell
close to the ideal -1°C per minute (Mr. Frosty, Nalgene No. viability may decline due to the DMSO.
5100-0001; or StrataCooler, Stratagene No. 400005). Alternately, 6. Place the vials into a pre-cooled (4°C), controlled-rate freeze
the vials can be placed into a polystyrene box with 15 mm (3/4 chamber and place the chamber in a mechanical freezer at
inch) thick walls and 1 litre capacity packed with paper, cotton -70°C (or colder) for at least 24 hours. Alternately, use a
wool, or foam peanuts for insulation. precooled (4°C) programmable freezer unit set to cool the
vials at -1°C per minute until a temperature below -40°C
Liquid nitrogen freezer storage is achieved and then set to abruptly drop to -130°C.
7. Quickly transfer the vials to a liquid nitrogen or -130°C
The ultra-low temperatures (below -130°C) required for long-term freezer. Frozen material will warm up at a rate of 10°C per
storage can be maintained by specialised electric freezers or minute and cells will deteriorate rapidly if warmed above
more commonly by liquid nitrogen freezers. There are two basic -50°C.
types of liquid nitrogen storage systems: immersing vials in the 8. Record the location and details of the freeze.
liquid and holding vials in the vapour phase above the liquid. 9. After 24 hours at -130°C, remove one vial, restore the cells
The liquid-phase system holds more nitrogen and thus requires in culture medium, and determine their viability and sterility.
less maintenance. However, there is always a chance that
some liquid will enter improperly sealed vials which may Recovery of cryopreserved cells
explode when retrieved. For this reason ATCC strongly
recommends storage in vapour-phase systems. The cell solution in the frozen vial needs to be warmed as rapidly
as possible and then immediately combined with complete
Vapour-phase systems create a vertical temperature gradient culture medium and seeded into an appropriate flask. While cells
within the container. The temperature in the liquid nitrogen at grown in monolayers can be recovered from cryopreservation in
the bottom will be -196°C, whereas the temperature at the top multiwell plates, the results are not as consistent as with flasks.
will vary depending upon the amount of liquid nitrogen at the
bottom as well as the amount of time the container is opened. Some cell lines, such as hybridoma cultures, take several
To ensure safe storage of cells, be sure to keep enough liquid days before they fully recover from cryopreservation. Some
nitrogen in the container so that the temperature at the top is hybridomas show low viability on the first day in culture and
-130°C or lower. All storage systems should be equipped with will generate cellular debris. Viability for most cells declines and
temperature alarms. reaches a nadir at 24 hours post thaw. Most, if not all, of this
decline appears to be due to apoptosis (as opposed to
Cryopreservation procedure necrosis) induced by the stress of the cryopreservation process.22
After this time point, cells begin to recover and enter
The procedure below will work for most cell cultures and should exponential growth.
be modified as needed. Freeze medium formulations for all
ATCC cell lines are provided on the Product Information Sheet. 1. Prepare a culture vessel (T-75 flask) so that it contains at
Harvest cells in exponential growth. least 10 ml of the appropriate culture medium equilibrated
1. Check your cell culture for contamination from bacteria, for temperature and pH.
fungi, mycoplasma, and viruses (see Contamination, page 2. Remove the vial from the liquid nitrogen freezer and thaw
22) immediately before cryopreservation. In most cases, by gentle agitation in a 37°C water bath (or a bath set at the
the results of the contamination screen will be available some normal growth temperature for that cell line). Thaw rapidly
time after the cultures are cryopreserved (10 to 14 days). If until ice crystals have melted (approximately 2 minutes).
contamination is confirmed, then destroy the frozen material. 3. Remove the vial from the water bath and decontaminate it
2. Prepare a freeze medium consisting of complete growth by dipping in or spraying with 70% ethanol. Follow strict
medium and 5% DMSO (ATCC® No. 4-X). Do not add aseptic conditions in a laminar flow tissue culture hood for
undiluted DMSO to a cell suspension as dissolution of DMSO all further manipulations.
in aqueous solutions gives off heat.
Cryopreservation
4. Unscrew the top of the vial and transfer the contents to a

sterile centrifuge tube containing 9 ml complete growth
centrifugation (10 minutes at 125 x g). Discard the

medium. Remove the cryoprotectant agent by gentle
supernatant, taking care not to disturb the soft pellet, and

resuspend the cells in 1 or 2 ml of complete growth medium.
Pipette gently to loosen the pellet and break apart clumps.
(If the cells normally grow as clusters, avoid over-pipetting
during resuspension.) Transfer the cell suspension into the
medium in the culture vessel and mix thoroughly.
5. Examine the cultures after 24 hours and subculture as
needed.
Contamination
Contamination of cells in culture can arise from many sources antibiotic for treatment. It is best to test the contaminating
including other cell lines, reagents, supplies such as pipettes and microbe for its antibiotic sensitivity prior to treatment; this
culture vessels, equipment such as tissue culture hoods and allows for a shorter treatment time and limits exposure of the
incubators, and laboratory personnel. While the potential for cell line to potentially damaging reagents.
contamination is constant, the risk can be reduced or eliminated
by proper precautions: using only reagents of known quality and The cells are cultured for 1 to 2 weeks in the presence of the
sterility, quarantining new cell lines until they are tested to be free antibiotic, and then cultured without antibiotic for 1 to 2 months.
from contamination, performing routine maintenance and cleaning At this point, the line should be retested with a very sensitive test
of all equipment, and properly training cell culture personnel. method to make sure that the culture is clean. Periodic retesting
should be employed to make sure that the contaminant does not
Checking for microbial contamination reappear. Since antibiotics may be toxic to cells, a selected
When most bacterial contamination occurs, it usually occurs population that no longer exhibits qualities of the parental line
within a few days and is typically obvious to the naked eye: may result. It may be necessary to examine the cured culture to
Distinct changes to the medium such as turbidity, presence of determine if it is sufficiently similar to the original line.
particles visible in suspension, and a rapid decline in pH
(yellow colour, indicating acidity) are all indicators of bacterial Cellular cross-contamination
contamination. Fastidious bacteria species that grow very slowly
can be difficult to detect. Cross-contamination of one cell line with another can sometimes
lead to the replacement of the original cell with the contaminant,
Fungal contaminants may or may not cause a change in the pH particularly when the contaminant grows faster than the original
of the medium and can be distinguished from bacteria by line. HeLa cells are perhaps the most famous example of a
checking for the presence of filamentous structures in the cross-contaminating cell line overtaking and then masquerading
suspension. Yeast cells are larger than bacteria, but may not as the original.
appreciably change the pH of the medium, and will appear as
separate round or ovoid particles. In the 1950s and 1960s, many continuous lines were
unknowingly cross-contaminated with other cell lines including
Microbial media which can be used to test for bacterial and HeLa cells. In the 1970s and 1980s, as many as one in three
fungal contamination include blood agar, thioglycollate broth, cell lines deposited in cell repositories were imposters.26 This
tryptic soy broth, BHI broth, Sabouraud broth, YM broth, and cross-contamination was only uncovered with the development
nutrient broth with 2% yeast extract.23 However, some microbial of suitable genetic markers beginning in 1967.27 Indeed, several
contamination is not apparent. For example, the use of antibiotics “unique“ cell lines in ATCC’s collection turned out to be HeLa
can suppress bacterial growth and thus mask contamination. cells upon further study. Despite the confirmation of their HeLa
Some viral infections do not alter the morphology of the cells, and cell origin, cytogenetic analysis suggests that there are
detection of mycoplasma contamination requires specific assays. differences among these HeLa-derived cell lines. Several of
them possess unique properties. However, these cell lines
Mycoplasma contamination should not be used as functional models of their claimed
tissues of origin.
Cell lines are screened for mycoplasma contamination by direct
(agarose and broth culture) and indirect (Hoechst) methods.24,25 More recently, ATCC and other cell repositories have used DNA
For example, the fluorochrome Hoechst DNA stain will bind to polymorphisms in addition to enzyme polymorphisms, HLA
the DNA of mycoplasma and the organisms can be detected typing, and karyotyping to confirm the identity of their cell lines.
easily when examined using a microscope equipped with One of the most reliable methods to study DNA polymorphisms
appropriate fluorescence optics. The direct culture method is the profiling of short tandem repeats (STR) by PCR
requiring both broth and agar will permit isolation of cultivable amplification followed by capillary electrophoresis.28 STR profiles
strains as apparent by appearance of characteristic mycoplasma for all ATCC human cell lines are available on the website in the
colonies on the agar medium. catalogue descriptions.
Both direct and indirect methods for detection of mycoplasma Routine testing
are used at ATCC several times while a cell line is expanded
for the preparation of the token, seed and distribution stocks. Test cell cultures on a regular basis to ensure the absence of
contamination from both microorganisms as well as from other
Treating for microbial contamination cell lines. If contamination is found, discard the culture and
start fresh with a new stock.
Eliminating contamination from a cell line is time consuming and
does not always work. Discarding the culture and starting over
is preferred. However, if the cells are unique and irreplaceable,
one should first identify the contaminant and select a suitable
Biosafety
The need for precautions when experimenting with cells in culture

depends upon the source and nature of the biological material,
the experimental procedure, and the laboratory/containment
conditions. Since every situation is different, the risks need to
be identified and appropriate precautions need to be taken
before any work begins.
More information on risk assessment and precautions can be

found in the Center for Disease Control (CDC) publication
Biosafety in Microbiological and Biomedical Laboratories, (BMBL)
5th Edition.29 The text of this publication is available in its
entirety online (www.cdc.gov/biosafety/publications/). Information
on agent risk assessment and a description of the four biosafety
levels can be found in this publication.
ATCC assigns a biosafety level (BSL) to each cell line for

purposes of packaging for safe shipment. When a cell line is
known to contain an etiologic agent, ATCC classification is at
least comparable to the BSL assigned to the agent by the CDC
and in some cases the ATCC designation is more restrictive.
ATCC follows federal biosafety guidelines and takes several
factors into consideration when assessing potential hazard.
Biosafety Level 1
• Cell lines with animal origin not included under Biosafety

level 2
Biosafety Level 2
• Cell lines that harbor mycoplasma or any other BSL 2 agent*

• Cell lines exposed to or transformed by a primate
oncogenic virus
• Primate cell lines that contain viruses
• Cell lines carrying a part of certain viral genomes, even if
whole virus is not released from the cell30
As the recipient of a cell line, take into account not only the
nature of the material but also the manipulations employed
during its handling when assessing the potential laboratory risk.
For example, procedures involving large volumes of cell lines
that contain HIV or that include manipulation of HIV in high
concentration should be conducted under BSL 3 conditions.29
Note: It is not possible to screen cell lines for the presence of

every agent. For added precaution, ATCC handles all cell lines
under BSL 2 practices, even those classified as BSL 1.
It is prudent to treat all mammalian cell lines as potentially
hazardous.
*Some patent cell lines at ATCC are known to be contaminated

with mycoplasma and are noted as such in the catalogue.
Glossary
The following glossary was originally published by the Tissue Cell strain. A cell strain is derived either from a primary culture
Culture Association Terminology Committee in 1990.31 or a cell line by the selection or cloning of cells having specific
Anchorage-dependent cells or cultures. Cells, or cultures features must be defined. The terms finite or continuous are to
properties or markers. In describing a cell strain, its specific
the term strain will suffice. In any published description of a cell

derived from them, which will grow, survive, or maintain be used as prefixes if the status of the culture is known. If not,
function only when attached to a surface such as glass or
plastic. The use of this term does not imply that the cells are strain, one must make every attempt to publish the
normal or that they are not neoplastically transformed. characterisation or history of the strain. If such has already
Aneuploid. The situation in which the nucleus of a cell does not

been published, a reference to the original publication must be
made. In obtaining a culture from another laboratory, the proper
contain an exact multiple of the haploid number of designation of the culture, as originally named and described,
chromosomes, one or more chromosomes being present in must be maintained and any deviations in cultivation from the
greater or lesser number than the rest. The chromosomes may original must be reported in any publication.
Chemically defined medium. A nutritive solution for culturing

or may not show rearrangements.
Aseptic technique. Procedures used to prevent the introduction cells in which each component is specifiable and, ideally, is of
of fungi, bacteria, viruses, mycoplasma, or other microorganisms known chemical structure.
Clone. In animal cell culture terminology, a population of cells

in cell, tissue, and organ cultures. Although these procedures
are used to prevent microbial contamination of cultures, they
homogeneous and therefore the terms clone and cloned do not

also prevent cross-contamination of cell cultures as well. derived from a single cell by mitoses. A clone is not necessarily
Attachment efficiency. The percentage of cells plated (seeded, indicate homogeneity in a cell population, genetic or otherwise.
Cloning efficiency. The percentage of cells plated (seeded,

inoculated) which attach to the surface of the culture vessel
within a specified period of time. The conditions under which
such a determination is made should always be stated. inoculated) that form a clone. One must be certain that the
Autocrine cell. In animals, a cell which produces hormones, this term. (See colony forming efficiency.)
colonies formed arose from single cells in order to properly use
expresses the corresponding receptors. (See also endocrine Colony forming efficiency. The percentage of cells plated
growth factors, or other signalling substances for which it also
and paracrine.) (seeded, inoculated) that form a colony.
Cell culture. Term used to denote the maintenance or cultivation Contact inhibition of locomotion. A phenomenon characterising
of cells in vitro including the culture of single cells. In cell certain cells in which two cells meet, locomotory activity
cultures, the cells are no longer organised into tissues. diminishes and the forward motion of one cell over the surface
Cell generation time. The interval between consecutive

of the other is stopped.
divisions of a cell. This interval can best be determined, at Continuous cell culture. A culture which is apparently capable
synonymous with population doubling time.

present, with the aid of cinephotomicrography. This term is not of an unlimited number of population doublings, often referred
express the characteristics of in vitro neoplastic or malignant

to as an immortal cell culture. Such cells may or may not
Cell hybridisation. The fusion of two or more dissimilar cells transformation. (See also immortalisation.)
Crisis. A stage of the in vitro transformation of cells. It is

leading to the formation of a synkaryon.
Cell line. A cell line arises from a primary culture at the time of characterised by reduced proliferation of the culture, abnormal
the first successful subculture. The term implies that cultures mitotic figures, detachment of cells from the culture substrate,
culture. The terms finite or continuous are used as prefixes if

from it consist of lineages of cells originally present in the primary and the formation of multinucleated or giant cells. During this
the status of the culture is known. If not, the term line will suffice.
massive cultural degeneration, a small number of colonies
The term continuous line replaces the term established line. In an apparent unlimited in vitro lifespan. This process was first
usually, but not always, survives and gives rise to a culture with
virus (SV40). See also cell line, in vitro transformation, and in

any published description of a culture, one must make every described in human cells following infection with an oncogenic
vitro senescence.
attempt to publish the characterisation or history of the culture.
If such has already been published, a reference to the original
Cryopreservation. Ultra-low temperature storage of cells,

publication must be made. In obtaining a culture from another
laboratory, the proper designation of the culture, as originally
named and described, must be maintained and any deviations tissues, embryos, or seeds. This storage is usually carried out
in cultivation from the original must be reported in any publication. using temperatures below -100°C.
Glossary
Density-dependent inhibition of growth. Mitotic inhibition Histiotypic. The in vitro resemblance of cells in culture to a
correlated with increased cell density. tissue in form, function, or both. For example, a suspension of
Differentiated. Cells in culture that maintain all or much of the

fibroblast-like cells may secrete a glycosaminoglycan-collagen
specialised structure and function typical of the cell type in vivo.

matrix and the result is a structure resembling fibrous connective
be used along with culture. Thus, a tissue culture system

tissue, which is, therefore, histiotypic. This term is not meant to
Diploid. The state of the cell in which all chromosomes, except demonstrating form and function typical of the cells in vivo
sex chromosomes, are two in number and are structurally would be said to be histiotypic.
Homokaryon. A cell possessing two or more genetically identical

identical with those of the species from which the culture was
derived.
Electroporation. Creation by means of an electrical current of

nuclei in a common cytoplasm, derived as a result of cell-to-cell
fusion.
Hybridoma. The cell which results from the fusion of an

transient pores in the plasmalemma usually for the purpose
of introducing exogenous material, especially DNA, from the
medium. antibody-producing tumor cell (myeloma) and an antigenically
Embryo culture. In vitro development or maintenance of

stimulated normal plasma cell. Such cells are constructed
because they produce a single antibody directed against the
isolated mature or immature embryos. antigen epitope which stimulated the plasma cell. This antibody
Embryogenesis. The process of embryo initiation and

is referred to as a monoclonal antibody.
development. Immortalisation. The attainment by a cell culture, whether by
Endocrine cell. In animals, a cell which produces hormones,

perturbation or intrinsically, of the attributes of a continuous
cell line. An immortalised cell is not necessarily one which is
growth factors or other signalling substances for which the neoplastically or malignantly transformed.
located at a distance. (See also autocrine or paracrine.) In vitro senescence. The inability of a vertebrate cell culture
target cells, expressing the corresponding receptors, are
Epithelial-like. Resembling or characteristic of, or having the

to grow beyond a finite number of population doublings.
Neither invertebrate nor plant cell cultures exhibit this property.
In vitro transformation. A heritable change occurring in cells in

form or appearance of, epithelial cells. In order to define a cell
as an epithelial cell, it must possess characteristics typical of
epithelial cells. Often one can be certain of the histologic origin culture, either intrinsically or from treatment with chemical
and/or function of the cells placed into culture and, under these carcinogens, oncongenic viruses, irradiation, transfection with
conditions, one can be reasonably confident in designating the oncogenes, etc., which leads to the acquisition of altered
properties. This expression is distinguished from in vitro

cells as epithelial. The individual reporting on such cells should morphological, antigenic, neoplastic, proliferative, or other
use as many parameters as possible in assigning this term to a
to use the term epithelial-like.

culture. Until a rigorous definition is possible, it is more correct neoplastic transformation in that the alterations occurring in the
cell population may not always include the ability of the cells to
Euploid. The situation in which the nucleus of a cell contains

produce tumors in appropriate hosts. The type of transformation
should always be specified in any description.
Organ culture. The maintenance or growth of organ primordia

exact multiples of the haploid number of chromosomes.
Feeder layer. A layer of cells (usually irradiated or mitomycin-C or the whole or parts of an organ in vitro in a way that may allow
treated) that are nondividing but metabolically active, upon differentiation and preservation of the architecture and/or
which a fastidious cell type is cultured. function.
Finite cell culture. A culture which is capable of only a limited Paracrine. In animals, a cell which produces hormones, growth
proliferation. (See in vitro senescence.)

number of population doublings after which the culture ceases factors or other signalling substances for which the target cells,
vicinity, or in a group adjacent to it. (See also autocrine and

expressing the corresponding receptors, are located in its
Heterokaryon. A cell possessing two or more genetically endocrine.)
Passage. The transfer or transplantation of cells, with or without

different nuclei in a common cytoplasm, usually derived as a
result of cell-to-cell fusion.
Heteroploid. A culture whose cells contain chromosome

dilution, from one culture vessel to another. It is understood that
any time cells are transferred from one vessel to another,
number other than the diploid number. This is a term used only a certain portion of the cells may be lost, and therefore dilution
synonymous with subculture.

to describe a culture and is not used to describe individual of cells, whether deliberate or not, may occur. This term is
cells. Thus, a heteroploid culture would be one which contains
aneuploid cells.
Glossary
Passage number. The number of times the cells in the culture Undifferentiated. With animal cells, this is the state wherein the
the cell type in vivo.

have been subcultured or passaged. In descriptions of this cell in culture lacks the specialised structure and/or function of
process, the ratio or dilution of the cells should be stated so
that the relative cultural “age” can be ascertained.
Plating efficiency. This term originally encompassed the terms

attachment efficiency, cloning efficiency, and colony forming
efficiency; it is now better to use one or more of them in its place
because plating is not sufficiently descriptive. (See attachment
efficiency, cloning efficiency, and colony forming efficiency.)
Population density. The number of cells per unit area or volume

of a culture vessel, or the number of cells per unit volume of
medium in a suspension culture.
Population doubling level. The total number of population

doublings of a cell line or strain since its initiation in vitro. This
term is synonymous with cell generation time.
Population doubling time. The interval, calculated during the

logarithmic phase of growth in which cells double in number;
is not synonymous with cell generation time.

for example, 1.0 x 106 cells increase to 2.0 x 106 cells. This term
Primary culture. A culture started from cells, tissues, or organs

taken directly from organisms. A primary culture may be
time. It then becomes a cell line.

regarded as such until it is successfully subcultured for the first
Pseudodiploid. The condition in which the number of

chromosomes in a cell is diploid but, as a result of
chromosomal rearrangements, the karyotype is abnormal and
linkage relationships may be disrupted.
Saturation density. The maximum cell number attainable, under

specified culture conditions, in a culture vessel. This term is
usually expressed as the number of cells per square
centimeter in a monolayer culture or the number of cells per
cubic centimeter in a suspension culture.
Suspension culture. A type of culture in which cells, or

aggregates of cells, multiply while suspended in liquid medium.
Synkaryon. A hybrid cell which results from the fusion of the

nuclei it carries.
Tissue culture. The maintenance or growth of tissues in vitro in

a way that may allow differentiation and preservation of the
architecture and/or function.
Transfection. The transfer, for the purpose of genomic

integration, of foreign DNA into cells in culture. The traditional
microbiological usage of this term implied that the DNA being
transferred was derived from a virus. The definition as stated
here describes the general transfer of DNA irrespective of its
source.
Formulations of media not available from ATCC
There are cell lines in the collection that require media which Other Components
are not currently sold by ATCC. Some media may require the Glucose.........................................................................900.0
addition of serum or other supplements. Refer to the Product Phenol red........................................................................ 5.0
Ham’s MCDB 105 and 107

Information Sheet for specific recommendations for each cell line.
ACL-4 The formulation for MCDB 107 is given below. For MCDB 105,
L-Amino Acids
A medium for the cultivation of human tumor cell lines with or change glycine to 7.51 mg/litre and omit KCl.
without serum.32 It consists of a 1:1 mixture of RPMI 1640 or mg/l
Ham’s F12 or F12K and Dulbecco’s Modification of Eagle’s Alanine..............................................................................8.9
Medium plus the following: Arginine HCl..................................................................
Insulin...................................................................... 20 µg/ml 211.7
Transferrin..................................................................10 µg/ml Asparagine · H2O............................................................ 15.0
Sodium selenite........................................................... 25 nM Aspartic acid...................................................................13.3
Hydrocortisone............................................................ 50 nM Cysteine HCl · H2O........................................................... 8.8
Epidermal growth factor............................................. 1 ng/ml Glutamic acid..................................................................14.7
Ethanolamine...............................................................10 µM Glutamine..................................................................... 365.3
Phosphorylethanolamine ............................................10 µM Glycine............................................................................ 22.5
Triiodothyronine......................................................... 100 pM Histidine HCl · H2O......................................................... 21.0
Bovine serum albumin.............................................. 2 mg/ml Isoleucine..........................................................................3.9
4-(2-Hydroxyethyl)-1-piperazine- Leucine........................................................................... 13.1
ethanesulphonic acid buffer.....................................10 mM Lysine HC....................................................................... 36.5
Sodium pyruvate........................................................0.5 mM Methionine........................................................................ 4.5
Glutamine..................................................................... 2 mM Phenylalanine................................................................... 5.0
Eagle’s Basal Medium (BME)

Proline.............................................................................34.5
Serine............................................................................. 10.5
A simple synthetic medium in routine use. Not adequate for Threonine........................................................................11.9
L-Amino Acids
more fastidious cell types.34,35 Tryptophan........................................................................2.0
mg/l Tyrosine, 2Na · 2H2O........................................................ 7.8
Arginine...........................................................................17.5
Vitamins
Valine..............................................................................11.7
Cystine............................................................................12.0
Glutamine..................................................................... 292.0 d-Biotin..........................................................................0.007
Histidine.......................................................................... 7.75 D-Ca pantothenate......................................................... 0.24
Isoleucine........................................................................26.0 Choline chloride.............................................................. 14.0
Leucine........................................................................... 26.0 Folinic acid, calcium salt.............................................0.0006
Lysine ............................................................................ 29.0 i-Inositol.......................................................................... 18.0
Methionine........................................................................ 7.5 Niacinamide.................................................................... 6.11
Phenylalanine................................................................. 16.0 Pyridoxine HCl................................................................ 0.06
Threonine....................................................................... 24.0 Riboflavin........................................................................ 0.11
Tryptophan........................................................................4.0 Thiamine HCl..................................................................0.34
Tyrosine.......................................................................... 18.0
Inorganic Salts
Vitamin B12.................................................................. 0.136
Vitamins
Valine.............................................................................. 23.0
CaCl2 (anhyd)..............................................................110.99
Biotin...............................................................................0.24 KCl................................................................................ 149.1
Choline............................................................................0.12 KH2PO4........................................................................408.27
Folic acid........................................................................ 0.44 MgSO4 (anhyd)........................................................... 120.38
Nicotinamide................................................................... 0.12 NaCl..........................................................................6546.00
Pantothenic acid............................................................. 0.20 CuSO4 · 5H2O............................................................0.00025
Pyridoxal HCl.................................................................. 0.20 FeSO4 · 7H2O................................................................ 1.390
Riboflavin........................................................................ 0.04 MnSO4 · 5H2O........................................................... 0.00024
Inorganic Salts
Thiamine HCl..................................................................0.34 (NH4)6Mo7O24 · 4H2O...................... ........................... 0.00124
NiCl2 · 6H2O...............................................................0.00012
NaCl............................................................................5845.0 H2SeO3...................................................................... 0.00387
KCl................................................................................ 373.0 Na2SiO3 · 9H2O.............................................................. 0.142
Na2HPO4 · H2O............................................................. 138.0
SnCl2 · 2H2O..............................................................0.00011
MgCl2 · 6H2O................................................................. 102.0
NH4VO3......................................................................0.00059
CaCl2.............................................................................111.1
NaHCO3...................................................................... 1680.0
Formulations of media not available from ATCC
Other Components
ZnSO4 · 7H2O................................................................ 0.144 Leucine........................................................................... 50.0
Lysine HCl.................................................................... 240.0
Adenine HCl................................................................... 1.72 Methionine...................................................................... 50.0
Linoleic acid................................................................0.0028 Phenylalanine................................................................. 50.0
DL-a-Lipoic acid..........................................................0.0021 Proline.............................................................................50.0
Putrescine 2HC........................................................ 0.00016 Threonine........................................................................75.0
Thymidine................................................................... 0.0727 Tryptophan......................................................................40.0
Glucose.......................................................................720.64 Tyrosine.......................................................................... 40.0
Vitamins
HEPES......................................................................7149.00 Valine.............................................................................. 65.0
Phenol red, sodium salt................................................1.242
Adjust pH to 7.6. Use 2% CO2.

Sodium pyruvate...........................................................110.0 Ascorbic acid.................................................................. 17.5
Biotin...............................................................................0.02
HITES
Ca-pantothenate...............................................................1.0
Choline HCl.................................................................. 250.0
A medium for the selective cultivation of small cell lung Cyanocobalamin (Vitamin B12)........................................ 0.2
carcinomas, adenocarcinomas, and tumors from other organ Folic acid.......................................................................... 0.4
sites with and without serum.36 It can be formulated using either m-Inositol.......................................................................... 1.0
RPMI 1640 or a 1:1 mixture of DMEM: F-12K and supplemented Nicotinamide..................................................................... 1.0
with the following components: Pyridoxine HCl.................................................................. 1.0
Insulin ....................................................................... 5 µg/ml Riboflavin.......................................................................... 1.0
Inorganic Salts
Transferrin................................................................10 µg/ml Thiamine HCl..................................................................10.0
Sodium selenite.................................................3.0 x 10-8 M
Hydrocortisone...................................................1.0 x 10-8 M NaCl............................................................................6000.0
-Estradiol..........................................................1.0 x 10-8 M KCl................................................................................ 150.0
HEPES........................................................................10 mM Na2HPO4..................................................................... 300.0
L-Glutamine.................................................................. 2 mM KH2PO4..........................................................................80.0
Mitsuhashi and Maramorosch Medium for Insect

MgCl2 · 6H2O................................................................. 240.0
Tissue Culture
MgSO4 · 7H2O............................................................... 200.0
CaCl2 · 2H2O..................................................................120.0
Other Components
M and M medium is no longer available commercially. Consult NaHCO3...................................................................... 2240.0
Inorganic Salts
reference 37 for additional information on this formulation.
mg/l Glucose.......................................................................5000.0
CaCl2 · 2H2O................................................................ 200.0 Hypoxanthine.................................................................. 25.0
KCl................................................................................ 200.0 Glutathione..................................................................... 15.0
MgCl2 · 6H2O................................................................. 100.0 Phenol red...................................................................... 10.0
NaCl............................................................................7000.0
NaHCO3.........................................................................120.0
Other Components
NaH2PO4 · H2O.............................................................. 200.0
D-Glucose...................................................................4000.0
Lactalbumin hydrolysate............................................. 6500.0
Adjust pH to 6.5 with 2M KOH.

Yeastolate................................................................... 5000.0
Waymouth’s MB 752/1
Capable of supporting growth of several cell lines in the
L-Amino Acids
absence of serum.34,38
mg/l
Arginine HCl................................................................... 75.0
Aspartic acid...................................................................60.0
Cysteine HCl...................................................................90.0
Cystine............................................................................15.0
Glutamic acid................................................................150.0
Glutamine..................................................................... 350.0
Glycine............................................................................ 50.0
Histidine HCl.................................................................150.0
Isoleucine........................................................................25.0
References
1.
Hayflick L and Moorhead PS. Exp. Cell Res. 25: 585-621, 33. Macy ML and Shannon JE. Preparation of medium
1961. ATCCCRCM30. TCA Manual 3: 617-622, 1977.
2. Rubin H. Mech. Aging Devel. 98: 1-35, 1997. 34. Morton HJ. In Vitro 6: 89-108, 1970.
3. Wright WE and Shay JW. Nat. Biotechnol. 20: 682-688, 2002. 35. Eagle H. Science 122: 501-504, 1955.
4. Capstick PB et al. Exp. Cell Res. 44: 119-128, 1966. 36. Carney DN et al. Proc. Natl. Acad. Sci. USA 78:3185-3189,
5. Osato et al. J. Immunology 124: 533-540, 1980. 1981.
6 McLimans WF. Chapter 5. In: Growth, Nutrition and 37. Mitsuhashi J. Adv. Cell Culture 2: 133-196, 1982.
Metabolism of Cells in Culture, Vol.1. Rothblat GH and 38. Waymouth C. J. Natl. Cancer Inst. 22: 1003-1017, 1959.
Cristofalo VJ, eds. New York: Academic Press; 1972.
7. Freshney RI. Chapter 8. In: Culture of Animal Cells, 5th ed.
New York: Wiley-Liss; 2005.
8. Shipman C. Proc. Soc. Exp. Biol. Med. 130: 305, 1969.
9. People CA et al. In Vitro 18: 755, 1982.
10. Spierenberg GT et al. Cancer Res. 44: 2253, 1984.
11. Zigler JS et al. In Vitro 21: 282, 1985.
12. Karmiol S. Development of serum free media. In: Master
JRW, ed. Animal Cell Culture, 3rd ed. Oxford: Oxford
University Press; 2000.
13. Jacoby WB and Pasten IH, eds. Chapter 7. In: Methods in
Enzymology: Cell Culture, Vol. 58. New York: Academic
Press; 1979.
14. Bolin SR et al. J. Virol. Methods 48: 211, 1994.
15. Hyclone, Inc. Art to Science 15(1): 1-6, 1996.
16. Rudnicki MA and McBurney MW. Teratocarcinomas and
Embryonic Stem Cells - A Practical Approach. Oxford: IRL
Press Ltd.; 1987: p. 75.
17. Weiss SA et al. Meth. Mol. Biol. 39: 65, 1995.
18. Gabridge MG. Vessels for Cell and Tissue Culture. In:
Setting Up and Maintenance of Tissue and Cell Cultures.
Shannon: Elsevier Scientific Publishers; 1985: pp. 1-19.
19. Balin A.K et al. Atmospheric Stability in Cell Culture
Vessels. In Vitro 12: 687-692, 1977.
20. Gey GO. Am. J. Cancer 17: 752-756, 1933.
21. Baust JM et al. Cell Preservation Technology 1: 17-31, 2002.
22. Baust JM et al. Cell Preservation Technology 1: 63-80, 2002.
23. Quality Control Methods for Cell Lines, 2nd edition.
Rockville, MD: ATCC; 1992.
24. Freshney RI. Culture of Animal Cells, 5th ed. New York:
Wiley-Liss; 2005.
25. Lincoln CK and Gabridge MG. Cell culture contamination:
Sources, consequences, prevention and elimination. In:
Mather JP and Barnes D, eds. Animal Cell Culture Methods.
San Diego: Academic Press; 1998: p. 49.
26. O’Brien SJ. Science 98: 7656-7658, 2001.
27. Gartler SM. NCI Monograph 26: 167-195, 1967.
28. Master JR. et al. Proc. Natl. Acad. Sci. USA 98: 8012-8017,
2001.
29. Biosafety in Microbiological and Biomedical Laboratories,
(BMBL) 5th Edition (HHS Publication No. (CDC) 93-8395.
U.S. Department of Health and Human Services, Centers
for Disease Control and Prevention and National Institutes
of Health; U.S. Government Printing Office: Washington DC;
2007). It is available in its entirety online at
http://www.cdc.gov/od/ohs/biosfty/bmbl5/bmbl5toc.htm.
30. Weiss RA. NCI Monograph 48: 183-189, 1978.
31. Schaeffer WI. In Vitro Cell. Dev. Biol. 26: 97-101, 1990.
32. Gazdar AF and Oie HB. Cancer Res. 46: 798-806 and
6011-6012, 1986.
Selected additional references
Effects of microbial contamination, cross-contamination and Effects of long-term culturing

misidentification
57. Behrens I et al. Do cell culture conditions influence the
39. Bubenik J. Cross-contamination of cell lines in culture. carriermediated transport of peptides in Caco-2 cell
Folia Biologica. 46 (5):163-164. (2000) monolayers? European Journal of Pharmaceutical
40. Buehring GC et al. Cell Line cross-contamination: how Sciences. 19 (5):433-442. (2003)
aware are mammalian cell culturists of the problems and 58. Briske-Anderson MJ et al. Influence of culture time and
how to monitor it? In Vitro Cellular and Developmental passage number on morphological and physiological
Biology. Animal; 40 (7): 211-215. (2004) development of Caco-2 cells. Proceedings of the Society for
41. Denecke J et al. Falsification of tetrazolium dye (MTT) Experimental Biology and Medicine. 214 (3):248-257.(1997)
based cytotoxicity assay results due to mycoplasma 59. Chang-Liu CM et al. Effect of passage number on cellular
contamination of cell cultures. Anticancer Research. response to DNA-damaging agents: cell survival and gene
19 (2A):1245-1248. (1999) expression. Cancer Letters. 26 (113):77-86. (1997)
42. Drexler HG et al. Mix-ups and mycoplasma: the enemies 60. Esquenet M et al. LNCaP prostatic adenocarncinoma cells
within. Leukemia Research. 26 (4): 329-333. (2002) derived from low and high passage numbers display
43. Drexler HG et al. False leukemia-lymphoma cell lines: An divergent responses not only to androgens but also to
update on over 500 cell lines. Leukemia.17 (2):416-426. retinoids. Journal of Steroid Biochemistry and Molecular
(2003) Biology. 62:391-399. (1997)
44. Garnick RL et al. Raw materials as a source of 61. Langeler EG et al. Effect of culture conditions on androgen
contamination in large-scale cell culture. Developments in sensitivity of the human prostate cancer cell line LNCaP.
Biological Standardisation. 93:21-29. (1998) Prostate. 23 (3):213-223. (1993)
45. Kagemann G et al. Impact of Mycoplasma hyorhinis 62. MacLeod RA et al. Identity of original and late passage
infection on L-arginine metabolism: differential regulation Dami megakaryocytes with HEL erythroleukemia cells
of the human and murine iNOS gene. Biological Chemistry. shown by combined cytogenetics and DNA fingerprinting.
386 (10):1055-1063. (2005) Leukemia. 11 (12):2032-2038. (1997)
46. Langdon SP et al. Cell culture contamination: an overview. 63. Riley SA et al. Active hexose transport across cultured
Methods in Molecular Medicine. 88:309-317. (2004) human Caco-2 cells: characterization and influence of
47. MacLeod RA et al. Widespread intraspecies cross- culture conditions. Biochimica et Biophysica acta. 1066
contamination of human tumor cell lines arising at source. (2):175-182. (1991)
International Journal of Cancer. 83 (4):555-563. (1999) 64. Sambuy Y et al. The Caco-2 cell line as a model of the
48. McGarrity GJ et al. Cell culture mycoplasmas. In: The intestinal barrier; influence of cell and culture-related
Mycoplasma, Vol. IV. Razin, S and Barile, MF, eds. New factors on Caco-2 cell functional characteristics. Cell
York: Academic Press, pp.353-390. (1985) Biology and Toxicology. 21:1-26. (2005)
49. Markovic O et al. Cell cross-contamination in cell cultures: 65. Vierck JL et al. Interpretation of cell culture phenomena.
the silent and neglected danger. In Vitro Cellular and Methods in Cell Science. 22 (1):79-81. (2000)
Developmental Biology. Animal. 34 (1):1-8. (1998) 66. Wenger SL et al. Comparison of established cell lines at
50. Masters JR. HeLa cells 50 years on: the good, the bad different passages by karyotype and comparative genomic
and the ugly. Nature Reviews. 2:315-319; (2002) hybridisation. Bioscience Reports. 24 (6):631-639. (2004)
51. Masters JR. Human Cell Cross-contamination Since 1983. 67. Yu H et al. Evidence for diminished functional expression
In Vitro Cellular & Dev Bio – Animal, 40:10-A (2004) of intestinal transporters in Caco-2 cell monolayers at high
52. Melcher R et al. SKY and genetic fingerprinting reveal a passages. Pharmaceutical Research. 14 (6):757-762. (1997)
crosscontamination of the putative normal colon epithelial
cell line. NCOL-1. Cancer Genetics and Cytogenetics. Other
151(1):84-87 (2005)
53. Mirjalili A et al. Microbial contamination of cell cultures: a 68. Hartung T et al. Good cell culture practice: ECVAM good
2-years study. Biologicals. 33 (2):81-85. (2005) cell culture practice task force report 1, ATLA 30:407-414
54. Thompson EW et al. LCC15-MB cells are MDA-MB-435: A (2002)
review of misidentified breast and prostate cell lines. 69. Hay RJ et al. Cell Line Preservation and Authentication in
Clinical & Experimental Metastasis. 21:535-541. (2004) “Animal Cell Culture,” J.R.W. Masters (ed.), J. Wiley, Inc.,
55. Wenzel U et al. Reconsidering cell line cross-contamination Oxford University Press, New York City, (2000)
in NCOL-1. Cancer Genetics and Cytogenetics. 70. Nardone RM. Eradication of Cross-contaminated cell lines:
163 (1):95-96. (2005) A call for action, Available at: http://www. Biotrac.com/pages/
56. No Authors listed. Contamination of cell lines – a conspiracy authentication.html. Accessed May 22, 2006.
of silence. Lancet Oncology. 2 (7):393. (2001)
ATCC requests that cell lines acquired from ATCC be referenced in scientific publications with the common name followed by the
ATCC catalogue number; e.g., NIH/3T3, ATCC® CRL-1658™
Why do cell lines need to be authenticated?
A growing number of high impact journals including biotechniques, Cancer * YOUR report will contain:
Research and Nature now require information on the authentication of any
human cell line used in a scientific study prior to accepting the submitted paper. ~ A summary page which allows you to review
There is also increasing evidence that funding bodies will require evidence of your results at a glance and prioritise any
cell line authentication as part of their grant application review process. Short cell lines which may need your attention.
Tandem Repeat (STR) profiling is the industry’s preferred method for human cell
~ A clearly laid out design with a single
line identification.
sample per page detailing;
LGC Standards has years of experience in STR profiling through our forensic ~ Your sample’s STR profile,
science activities combined with cell biology expertise, gained through our ~ Reference profile,
collaboration with ATCC®. This experience enables us to offer a unique, fully ~ Electropherogram,
supported Cell Line Authentication program. ~ Analysis and guidance notes.
Our Cell Line Authentication program, is supported by an expert team who are ~ Your report will adhere to definitions outlined
available to provide advice and support with any questions you may have from by the forthcoming ASN0002: Authentication
sample preparation through interpretation of results and their implications. of Human Cell Lines: Standardisation of
LGC Standards has access to thousands of cell line reference profiles. We STR profiling. This is expected to become
produce a clearly laid out comprehensive report which provides the analysis the industry standard for authentication of
of your cell sample*. human cell lines.
Authenticating your cells couldn’t be easier, follow our quick and simple steps:
Fill in order form Prepare cells Send us your samples We’ll get you
Customer friendly A range of acceptable An easy shipment
the profile!
order form sample types process
For sample shipment we offer the option of a Transport Buffer, the greener shipping alternative. Transport Buffer is a proprietary
technology from LGC, which eliminates the need and hassle of transporting cells on dry ice, saving you from expensive shipping
costs. It works by lysing the cell sample and stabilising the DNA allowing your samples to be shipped safely to our facility at room
temperature.
Whether you are starting a new project or have been culturing you cells for a while, get them authenticated and know what you’re
really working with. Contact your local sales office or visit the website for more information.
www.lgcstandards.com/authenticate
FAQ
FAQ
How often should w
we
e authenticate
authenticate our cell lines?
Increasingly
y, publishing
Increasingly, g and funding bodies are requesting
sting specific infor mation on the
information e authentication
authentication of any
any human cell line used in
researc h. The emerging
research. emergin
ng trend seems to be tha
thatt cells are
a required to be authentica ted
authenticatedd within 6 months prior to the da
ate of ar
date ticle
article
submission F
submission. or good llaboratory
For laborator y practice, it is ad
practice, d to authentica
vised
advised te a
authenticate att the beginning
ng and during a project rrather
ather than
han only a
att the
end. This ensures yyou
ou truly
truly know what
what you
you are working
workin
ng with.
W hat if I don’t
What don’t want
want to
to authenticate
authenticate my
my cells?
Man
Manyy jour nals still ha
journals ve no fformal
have or mal requirement for
for info mation on the authenticity of human
or
information h cell lines used in original
al researc h.
research.
Howeverr, we
However, we are seeing
seein
ng increasing numbers
numbers of researchers
researchers who have cchosen
have hosen jour als whic
na
journals whichh do not require authentication, o
authentication, nly tto
only o
have one of their re
have view
wers specifically request tha
reviewers thatt the
e cell line be authenticated.
authenticated. LGC
LGC
C Standards expects
expects this
this trend
trend to
to continue
continue and
and
believe that
believe that it will become
beco
ome increasingly difficult to publish
publish data
data generated using unaut
generated thenticated human cell lines
unauthenticated lines..
I’m working with a primary

working prrimar y cell culture
culture and I think there ar
there e multiple cell types
are types..
Can you tell me the types
you ty
ypes of cells I am working wi
working th?
with?
Cell Line Authentication
n ser
Authentication vice crea
service tes STR profiles whic
creates whichh are genetic
genetic fingerprints
fingerprints of the cell sample supplied. All cells
ls from
from an
an
individual (bar a ffew
individual ew unusual
un
nusual instances) have
have the same
sam
me genetic
genetic make-up. STR profilin
make-up. ng will ena
profiling ble the g
enable enetic origin
genetic n of the cell
sample to be identified
d, how
identified, ever it cannot determine
however deter mine the
t phenotype of the cells in the
e sample.
sample. Phenotypic ccharacterisation
haracterrisation tests
tests,,
suc
suchh as expression
expression of cell markers
markers would
would be required
d to answer this question.
answer
I ha
ave tw
have o cell lines which
two w h are
whic are recorded
recorded as being related,
related, however
however the rrecords
ecords ma
m y not be rreliable.
may eliable.
Can we deter
we mine if these
determine t were isola
were ted fr
isolated om the same indi
from vidual?
individual?
The STR profiles generrated from the tw
generated o cell samples can be compared and our analysis
two ysis team can advise
advise on the likely
likelyy relatedness
relatedness
of these cell samples
samples.. Examples
E inc lude; a cancer biopsied
include; psied a
att both the primary
primar y and a metastatic
metastatic sites; or a parental cell
ell line and
a sub-c lone/stable tr
sub-clone/stable ansf
sfectant.
transfectant.
W hat per
What centage of cell
percentage c lines are misidentified or contamina
are ted?
contaminated?
There have been a number
have mber of publica tions over
publications over recent years
years reporting
repor ting approximately
approximately 15-20% of human cell lines in culture
ulture are either
contamina ted or misidentified.
contaminated entified. Our experience aligns with these published figures
experience figures..
W hat if w
What e don
we ’t have
don’t have a reference
reference for
for our cell line?
?
We can still g
We enerate an
generate n STR profile and searc
searchh the database
da
atabase to see if your
your cell line matches
matches one of the common contaminants.
contaminants
taminants. If your
your
cell line doesn’t ma
doesn’t tch a cell in the da
match tabase, and the profile
database, p g
has good attributes it is reasonable
attributes reasona
easonable to conclude
conclude that
that it is a unique
q cell line.
line.
We do advise
We advise tha
thatt yyou
ou have all yyour
have our unidentified cell lines
es profiled a
att the same time to ensure that
that the STR profiles they
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ATCC Cell Culture

Starter culture Token freeze Seed stock Distribution

• Contamination checks • Contamination checks • Contamination checks

• Species verification • Species verification • Species verification

• Post-freeze recovery • Post-freeze recovery • Post-freeze recovery

• Post-freeze viability • Post-freeze viability • Post-freeze viability

• Growth curve • Characterisation tests • Characterisation tests

Authenticated Robust growth

Experimental success has been shown to correspond

To maintain high cell culture standards and ensure reliable,

ATCC authenticates cell lines routinely with the

I Short tandem repeat (STR) profiling establishes a DNA

fingerprint for human cell lines.

Figure 2 I STR profile of two unrelated cell lines. Top:

“Evidence suggests that up to one-third of tumor cell

processes. as well as variation within the cell line.

The ATCC COI assay is used to reliably determine the species

6 Getting started with an ATCC cell line

7 Cell growth and propagation

13 Complete growth media

18 Culture vessels and surfaces

28 Formulations of media not available

32 Why do cell lines need to be authenticated?

35 Material Transfer Agreement

39 The LGC Standards – ATCC Partnership

Getting started with an ATCC cell line

If the cells are attached and growing in a monolayer:

gentle centrifugation (10 minutes at 125 x g). Discard the

supernatant, and resuspend the cells in 1 or 2 ml of complete

Cell growth and propagation

higher split ratio. Passage number is generally the number of

of population doublings (or population doubling level, PDL)

As noted in the section on culture vessels, cell lines grow either

• Lag phase — Immediately after seeding of the culture

Cell growth and propagation

Calculate the population doubling time, or the time required for

a culture to double in number, with the following formula:

To ensure that the characteristics of your cell line remain Temperature

Cell growth and propagation

morphology of the cells. Bacterial contamination will appear as

Most adherent cells should be attached firmly to the surface. In

Cells in suspension culture grow either as single cells or as

numbers. Hemocytometers (also spelled hemacytometers) are

Cell growth and propagation

at 125 x g) and then resuspend the cells in 6 to 8 ml of

Cell growth and propagation

Cells are difficult to remove.

Cells form clumps after dissociation.

gentle centrifugation (10 minutes at 125 x g).

Cells have difficulty reattaching to the flask.

medium or centrifuge (5 minutes at 125 x g) the cells down

Viability is lower than expected.

Cell growth and propagation

Viability is lower than expected.

Adapting a monolayer cell line to grow in suspension

Some cell lines such as L-929 (ATCC® CCL-1™), HeLa (ATCC®

1. Dissociate the cell monolayer using standard procedures.

centrifugation (10 minutes at 125 x g). Count, and re-seed

a fresh flask with fresh medium at 2.5 x 105 cells/ml.

Complete growth media

Dulbecco’s Modified Eagle’s Medium (DMEM) has roughly

Complete growth media

DMEM/F12 Medium is a 1:1 mixture of Dulbecco’s modified